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With the rise of genomics, taxonomic identification practices have progressed towards the use of culture-independent methodologies, such as metagenomics. Nevertheless, it has recently been shown that the use of culture-dependent procedures not only helps discriminate and describe the microorganisms present but they are in fact invaluable as they grant information unobtainable otherwise. Furthermore, the combined use of both methodologies provides synergistic gains compared to the results obtained separately. Considering that these insights are still mostly unexploited in regard of fungal diversity, we present an evaluation of a culturomics-based approach to the study of the mycological biodiversity present in the outer layer of a semi-hard goat cheese with visible mould contamination. We have staged a two- pronged setup by metagenomics-based assessment of the Internal Transcribed Spacer region contents, and a culture-based evaluation through Sanger sequencing of the same region of a subset of the total fungal strains isolated in four culture media. We have obtained eight unique representative OTUs out of 177 isolates in the culture approach, whilst with metagenomics, out of nine ASVs obtained from bioinformatic sample treatment using Q2- ITSxpress, we have obtained five representative OTUs. Combining both results, a total 13 OTUs were identified, two being exclusive to metagenomics, three common to both methods, and five exclusive to the culture approach. Our results demonstrate that a combination of both methodologies provides better results in terms of total OTUs classified in comparison to each approach individually. Moreover, the culture-based approach provided a greater number of OTUs comparatively, as well as the number of unique OTUs phylogenetically classified., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, of LABBELS Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems (LA/P/0029/2020), and of MIRRI-PT (Pólo Norte) project (PINFRA04/84445/2020) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Microbial Culture Collections (CCs) are biobanks that preserve and provide microorganisms to the user communities. In the Portuguese landscape, CCs differ considerably regarding size, taxonomic target group, level of organization and implemented quality management systems. Since 2017, the CCs have been working together in the Portuguese microbiological Resource Centre Network (Pt-mBRCN), which was recognised as the Portuguese MIRRI node of the ESFRI Landmark MIRRI in June 2020, to promote a common voice, share experience and foster cooperation. As part of its operation, Pt-mBRCN/MIRRI- PT aims to guarantee the quality of its microbial holdings and associated data, and to implement common procedures and quality management systems to mitigate the current resource/data fragmentation microbial resources. To accomplish these objectives, a strong investment in dedicated human resources and cutting edges technologies is required, which can be partly achieved through competitive funding, such as MIRRI-PT-Polo Norte project. An example of activities within Pt-mBRCN is the collaboration between Micoteca da Universidade do Minho (MUM) and Azorean Regional Veterinary Laboratory Micoteca (LRV-Micoteca). This is providing conditions to accelerate LRV-Micoteca strains requalification process and catalogue improvement, allowing a faster development and establishment of this emerging CC, paving the future to implement ISO 17025 accreditation system., This work was supported by the European Unions Horizon 2020 Research and Innovation programme, under the Grant Agreement no 871129. This publication reflects only the authors view and the Commission is not responsible for any use that may be made of the information it contains., This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020, and of MIRRI-PT (Pólo Norte) project (PINFRA04/84445/2020) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

The demand for functional ingredients (such as prebiotics) has risen as an effect of increasing health consciousness. Amongst them, xylooligosaccharides (XOS) shows prebiotic effect at lower doses, in contrast with fructooligosaccharides (FOS), isooligosaccharides (IOS) or galactooligosaccharides (GOS). We have developed a process for obtaining XOS from wheat straw applying an alkali pre-treatment to the raw material followed by enzymatic hydrolysis with xylanases. Using thermophilic xylanases, the arabinoxylan extraction from the solid wheat straw was increased significantly compared to mesophilic counterparts. However, a neutralization (and washing) step of the pre-treated wheat straw was still needed before the enzymatic treatment. Therefore, finding thermophilic and alkaliphilic endo-xylanases would offer a substantial advantage for the overall optimization of XOS production. In order to increase the possibilities of finding endo-xylanases and beta-xylosidases in thermophilic and alkaliphilic environments able to act on wheat straw xylan, we carried out an enriched culture using wheat straw as inoculum and the sole C source and sterile Tinteiro thermal spring waters (Ourense) as solvent and source of mineral micronutrients. Tinteiro thermal waters are characterized by a high pH (8.6), moderate temperature (50 oC) and a high content in sulfur and carbonate compounds. Samples were taken periodically from the culture medium and were grown in xylan- agar plates. Positives strains were visualized with Congo-red stain and identified by 16S gene sequencing. Their full-length 16S genes were amplified and sequenced. We have obtained two promising candidates strains. Their 16S rRNA gene sequences exhibited similarity to genus Paenibacillus and Neobacillus. From the Paenibacillus strain, identified as P. barengoltzii, produces different lignocellulose degrading enzymes have been described. The Neobacillus strain was related to N. thermocopriae and N. sedimentimangrovi and presents highly promising enzymes. Endo-xylanase highlights for its ability to work at pH 7-10, with maximal activity at 10. Also, beta-xylosidase was detected which presumably shares alkaliphilic characteristics. Higher characterization of the enzymes should be performed prior to industrial XOS production. In conclusion, a microorganism characterized as Neobacillus sp. that produces endo-xylanases with extremophile characteristics was isolated from a non-extremophile environment incubating the inoculum at the desired conditions of the final application., Work supported by the Spanish Ministry of Science and Innovation (RTI2018-099249-B-I00) and Iacobus program 2020/2021, Xunta de Galicia, Spain (CITACA Strategic Partnership, Reference: ED431E 2018/07). Clara Fuciños is financed as Distinguished Researcher by the University of Vigo (Programa de Retención de Talento 2020). It was partially supported by FCT - UID/BIO/04469/2019 unit., info:eu-repo/semantics/publishedVersion

Mould growth on cheese is very common and the main contamination source is believed to be the environment in the production facilities. The growth of filamentous fungi on the cheese surface makes the product undesirable and can even present a health risk due to the production of secondary metabolites, such as mycotoxins. In fact, conditions at which ripening occurs are favourable to fungi development, and indigenous moulds of the ripening rooms may colonize the surface of cheeses. This work intended to investigate the air mycobiota of a Portuguese cheese industry to uncover the role of the air of the ripening chamber as the source of the observed cheese contamination. Air samples were collected using the impaction method on DRBC Petri dishes. Colonies were counted and, after seven days, the most common fungi were isolated in PDA medium. A contaminated cheese was also chosen to isolate visible fungi. Fungal isolates were identified through observation of microscopic characteristics and molecular methods (analysis of ITS or partial benA). Three isolates were retrieved directly from the cheese and seven from the air samples. The identified genera were Penicillium, Aspergillus, Botrytis and Cladosporium. From cheese, only one species of Penicillium was isolated, which corresponded to the most abundant species from the air samples. The most relevant genus in the air mycobiota was Penicillium, with 3 isolates from 3 species, whereas all other genera were only represented by one species. This study indicates that the air of the ripening room is a source of contamination for cheese and mitigation measures must be implemented., Teresa Vale Dias thanks for the Ph.D. scholarship given by the Foundation for Science and Technology (FCT) —2020.05849.BD. This study was also supported by FCT under the scope of the strategic funding of CEB (UIDB/04469/2020) and CIMO (UIDB/00690/2020)., info:eu-repo/semantics/publishedVersion

Ochratoxin A (OTA) is a well-studied mycotoxin that poses severe health risks. OTA is mainly produced by Aspergillus and Penicillium species associated with food spoilage and it is present in a wide diversity of food and feed products. Recent studies have reported the presence of OTA in food matrices where known OTA producers are not present1,2. For that reason, other species such as P. crustosum are now being considered. A recent study using comparative genomic analysis3 clarified the OTA biosynthetic gene cluster composition. In order to gain insight into the secondary metabolism of P. crustosum, this study aimed to sequence and explore the complete genome of strain MUM 16.125. This strain was isolated from cheese rind sample contaminated with OTA in which no known OTA producers were present1. The genome assembly comprises 199 contigs with a total length of 30.95 Mb and contains 10975 predicted protein-coding genes. In total, 109 gene clusters potentially related with secondary metabolism were identified, including putative gene clusters for penitrem, clavaric acid or naphthopyrones biosynthesis. Nevertheless, no evidence of an OTA biosynthetic gene cluster was found. A total of 83 complete and 49 partial protein sequences from published OTA biosynthetic genes from 11 Aspergillus and 3 Penicillium species were queried against the predicted P. crustosum proteins. Only 3 strong matches were found (to a short partial P. verrucosum PKS and 2 P. thymicola chloroperoxidases) but matches to complete key genes were absent. Considering these findings, it appears that strain MUM 16.125 lacks the most common genetic pathway to produce OTA, providing important information relevant to understand the role of P. crustosum as putative OTA producer. Nevertheless, the additional secondary metabolism gene clusters found (such as penitrem, clavaric acid or naphthopyrones) highlight the potential of this strain for metabolite production, including other mycotoxins or compounds with antioxidant, anticancer or antibiotic properties., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of CEB (UID/BIO/04469/2019) and iBiMED (UIDB/04501/2020) units; and by CANCYL (POCI-01-0145-FEDER-031849) and GenomePT (POCI-01-0145-FEDER-022184) projects, info:eu-repo/semantics/publishedVersion

[Excerpt] The Botryosphaeriaceae family includes genera frequently related with plant diseases. Nevertheless, human infections caused by fungal plant pathogens are becoming more frequent. Neoscytalidium species are mainly phytopathogenic but cases of phaeohyphomycosis have been reported. Within this genus. four species are currently recognized: N. dimidiatum, N. orchidacearum, N. novaehollandiae, and N. oculus. Although N. dimidiatum is frequently found in soil. it has been identified as an etiologic agent of onychomycosis or dermatomycosis. On the other hand, N. oculus was isolated and identified as an etiologic agent of an ocular lesion. All these species are very similar with regard to their macroscopic and microscopic traits, which lead to complex and imprecise identification based on morphological traits alone. [...]

[Excerpt] With the continuous growth of the world population, substantial increases in food production will be required as well as the need to guarantee its safety. Problems related to food/feed contamination have been frequently reported, including those related with fungi and their production of mycotoxins. Mycotoxins are fungal secondary metabolites that can cause severe health issues in either humans or domesticated animals when ingested, inhaled and/or absorbed. Ochratoxin A (OTA) is one of the most studied and poses a severe health risk. This toxin is present in a wide diversity of food and feed products with Aspergillus and Penicillium species being mainly associated with food spoilage and OTA contamination. The major OTA producers are P. verrucosum, P. nordicum and A. carbonarius. Recent studies have reported the presence of OTA in food matrices where known OTA producers are not present. Therefore, and based on previous evidences, other species, such as P. crustosum, are now being considered. The main goal of this work was to search for potential OTA producers among P. crustosum strains with different geographic origins and to search for potential genetic differences at the sub-species level. [...]

Objective Identify yeast and yeasts-like fungal species isolated from onychomycosis cases by partial sequencing of D1/D2 from 26S rDNA region. Methods Forty-four yeast and one like-yeast isolates from patients with onychomycosis were analyzed. The D1/D2 domain of the 26S rDNA region was amplified using the NL1/NL4 primers and subsequently sequenced. Obtained sequences were aligned using CLUSTAL W and phylogenetic analysis was carried out with MEGA v7 software. Bootstrap statistical support of at least 70% was considered to group samples. Results Phylogenetic analysis allowed the identification of 13 species: Candida parapsilosis (33.0%), C. tropicalis (15.5%), C. albicans (15.5%), C. haemulonis (7.0%), C. intermedia (7.0%), C. cabralensis (4.4%), C. orthopsilosis (2.2%), C. metapsilosis (2.2%), Debaryomyces hansenii (2.2%), Meyerozyma guilliermondii (4.4%), Pichia kudriavzevii (2.2%), Trichosporon asahii (2.2%), and Wickerhamomyces onychis (2.2%). Conclusion Candida parapsilosis continues to be the most prevalent yeast as etiologic agent of onychomycosis in Colombia. In the current study, it is important to highlight the isolation of C. cabralensis, usually recognized as an environmental yeast, and W. onychis. To our best knowledge, this is the first report of an onychomycosis case caused by the former species while the latter has only been associated with a single clinical case published in 1965 as Pichia onychis.

Las micotoxinas son metabolitos secundarios producidos por hongos filamentosos. La Ocratoxina A (OTA) y las Aflatoxinas (AFs) son micotoxinas altamente tóxicas y carcinógenas que se encuentran como contaminantes en alimentos para humanos y animales. El Capsicum (ají o pimiento) es un género de plantas, dicotiledóneas nativo de las regiones tropicales y subtropicales de América, pertenecientes a la familia de las solanáceas. En Chile, el cultivo de Capsicum annuum L. cv. Cacho de Cabra se extiende desde la Región de Arica y Parinacota hasta la Región de La Araucanía, correspondiendo a 9 de las 16 Regiones del país y cubriendo aproximadamente 522 hectáreas de área plantada. Las bayas del ají Cacho de Cabra se utilizan para la producción de un polvo rojo tradicional conocido como Merkén, que es un aliño picante con sabor ahumado. El Merkén es un producto intrínsecamente asociado con el grupo étnico ancestral Mapuche. Al igual que con cualquier otro producto agroalimentario, las bayas del ají Cacho de Cabra y el Merkén procesado también son susceptibles a la infección por hongos que pueden generar contaminación con micotoxinas. Cepas de Alternaria, Aspergillus, Cladosporium, Fusarium, Penicillium y Rhizopus son contaminantes comúnmente halladas en bayas de Capsicum y en sus productos derivados. Entre éstas, cepas de los géneros Aspergillus, Fusarium y Penicillium son las más importantes en términos de capacidad de producción de micotoxinas en dicha matriz alimenticia. En 2017, el Ministerio de Salud de Chile reportó dos casos de contaminación por Merkén con micotoxinas, principalmente OTA. Conocer los puntos de infección por hongos y de contaminación con micotoxinas en la cadena de producción del ají Cacho de Cabra y Merkén, sería una contribución para la implementación de buenas prácticas agroalimentarias, conduciendo a la solución de ese problema. Sin embargo, esa información no está todavía disponible. En ese contexto, el presente trabajo presentará los resultados del potencial micotoxigénico de la micobiota perteneciente a los géneros Aspergillus y Penicillium aislada en los diferentes puntos de la cadena de producción tradicional del ají Cacho de Cabra y del Merkén y posibles puntos de contaminación con OTA y AFs. Además, se discutirán las interacciones ecológicas entre los hongos micotoxigénicos y las bayas de Capsicum annuum L. cv. Cacho de Cabra y del Merkén., info:eu-repo/semantics/publishedVersion

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Berry fruits of Capsicum annuumcv. "Cacho de Cabra” are used for the manufacture of a traditional pepper powder known as Merkén, which is a Chilean spicy powder with smoky flavour produced through both industrial and artesian processes. Merkén contamination with Ochratoxin A (OTA) is a main concerning for industries and local producers. Therefore, the main goal of this study was to search for the cultivable mycobiota on the whole processing stages of chilli used in traditional Merkén production. Capsicum annuum samples were provided by 8 farmers from 4 localities of the Chilean Region of La Araucanía. Chilli berry fruits were collected at five different sampling time points of production: (I) just at the day of ripe fruits harvest; (II) after 30 days of harvest (drying process); (III) during the smoking process; (IV) ingredients added to Méken (e.g., coriander seeds) and (V) Merkén samples from local markets. Samples were used for isolation of cultivable mycobiota on MEA, DRBC and DG18 media. Isolated fungal strains were identified using classical morphology, MALDI-TOF MS and molecular biology. A total of 225 fungal strains belonging to 9 fungal genera were identified. From the sampling point I, 61 fungal strains were isolated. From these, strains were distributed among Penicillium spp. (25), Fusarium spp. (14), Alternaria spp. (9), Aspergillus spp. (7) and others (6). From the sampling point II, 46 fungal strains were isolated. These strains were distributed among: Penicillium spp. (24), Aspergillus spp. (12), Alternaria spp. (7) and other species (3). From the sampling point III, 85 fungal strains were identified. These strains were distributed among: Penicillium spp. (63), Aspergillus spp. (18), and other species (4). From the sampling point IV, were isolated only Penicillium spp. (6), Aspergillus spp. (4). While, from sampling point V, the isolated strains were Penicillium (10), Aspergillus (4), Fusarium (1), and other (8). This study demonstrates that Merkén production process is increasingly selective for occurrence of Aspergillus and Penicilliumspecies, which already emerge in the early stages of the Capsicum production. In addition, Alternaria and Fusariumwere also present in C. annuum, mainly in fresh fruits. The evaluation of OTA production by the potential OTA producers fungal species isolated in the whole Merkén production/commercialisation chain is now under course

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Portuguese Foundation for Science and Technology (FCT). It was under the scope of the strategic funding of the UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01–0145-FEDER-006684) and the BioTecNorte operation (NORTE-01–0145-FEDER-000004), funded by the European Regional Development Fund through Norte2020—Programa Operacional Regional do Norte (Portugal)

The 10th International Palestinian Conference of Laboratory Medicine and The 15th Arab Conference of Clinical Biology, Among certain groups of filamentous fungi that produce mycotoxins, relevant contaminates in food, the genus Penicillium is of great importance. Penicillium is ubiquitous in nature and inevitable, although it can be controlled from the field to the fork. Mycotoxins are fungal secondary metabolites that cause sickness or death in people when ingested, inhaled, and/or absorbed. Major mycotoxins associated with common penicillia are: Ochratoxin A (P. verrucosum and P. nordicum), patulin (P. expansum), citrinin (P. expansum), cyclopazonic acid (P. camemberti), penicillic acid (P. radicicola) and secalonic acid D, F (P. griseofulvum). Penicillia identification is time-consuming and sounder polyphasic identification, which includes phenotypic and genotypic approaches, is recommended. However, in many laboratories, the standard character for identification is still morphology. Taking this into account, results from Penicillium species isolated from Tunisian apples, Chilean traditional chilli (Merkén), Italian cheeses and their mycotoxin profiles (patulin and ochratoxin A) will be presented in this work. For morphological analyses, isolates were inoculated in triplicate in different media. Fungi grown in MEA for colony and microscopy analyses were used. Multilocus sequence analysis was performed through comparison of partial -tubulin, calmodulin and ITS with sequences available in GenBank. Specific primers for genes involved in the mycotoxins pathways were used for PCR amplification. After extraction the mycotoxins were quantified using HPLC-FLD (fluorescence detection). From Tunisian apples isolates, a novel species Penicillium tunisiense of section Ramosa is proposed. This is not a patulin producer with the idh gene negative in contrast with the other dominant P. expansum isolates. In addition, ochratoxigenic strains P. verrucosum and P. crustosum were isolated from chilli and cheese samples, respectively, and characterised with genes involved this mycotoxin production. Our findings show that mycotoxigenic Penicillium strains, as food contaminants, remain an important field of study and more knowledge needs to be learned., info:eu-repo/semantics/publishedVersion

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