Corresponding author: João Trovão (
Academic editor: C. Gueidan
When colonizing stone monuments, microcolonial black fungi are considered one of the most severe and resistant groups of biodeteriorating organisms, posing a very difficult challenge to conservators and biologists working with cultural heritage preservation. During an experimental survey aimed to isolate fungi from a biodeteriorated limestone art piece in the Old Cathedral of Coimbra, Portugal (a UNESCO World Heritage Site), an unknown microcolonial black fungus was retrieved. The isolated fungus was studied through a complete examination based on multilocus phylogeny of a combined dataset of ITS rDNA, LSU and
Trovão J, Tiago I, Soares F, Paiva DS, Mesquita N, Coelho C, Catarino L, Gil F, Portugal A (2019) Description of
Microcolonial black fungi (
Classical morphological approaches used to identify
In 2013, UNESCO recognized the University of Coimbra, Alta and Sofia (Coimbra, Portugal) as a World Heritage Site. Inside this area, several monuments exhibit clear signs of biodeterioration, including microcolonial black fungi proliferation. During one experimental survey performed in the Old Cathedral of Coimbra (Sé Velha de Coimbra), an unknown slow-growing microcolonial black fungi with late melanization was retrieved. Therefore, we aim to determine, through a multi-gene analysis (ITS rDNA, LSU and
The Old Cathedral of Coimbra is the only Portuguese Romanesque cathedral from the Reconquista times to have survived relatively intact until now. The Romanesque church is located on a hillside in the historic city center and was constructed between the 12th and early 13th centuries. The single-floored cloister is arranged laterally to the south of the church and is surrounded by five chapels carved in yellow dolomitic limestone. Samples were collected using sterile scalpels by scrapping small areas (3 cm2) into a collection tube, from a deteriorated art-piece in the Santa Maria chapel (
DNA from pure fungal cultures was obtained using the Extract-N-Amp Plant PCR Kit (Sigma-Aldrich, USA) with several modifications. A small portion of the colonies was scraped from the agar surface using a sterile scalpel, submerged in 10 µl of extraction solution and incubated in an ABI GeneAmp 9700 PCR System (Applied Biosystems, USA), with the following protocol: 65 °C for 10 min, followed by 95 °C for 15 min. After the incubation, reactions were stopped by adding 10 µl of elution solution. The obtained genomic DNA was subjected to PCR amplification with a final volume of 25 µl, with 12.5 µl of NZYTaq Green Master Mix (NZYTech, Portugal), 1 µl of each primer (10 mM), 9.5 µl of ultra-pure water and 1 µl of template DNA. Primer pairs ITS1-F/ITS4 (
DNA sequences were assembled using the Geneious R11.0.02 software (
To examine heat resistance, mycelia from grown cultures on PDA were homogenized and heated at 75 °C for 30 min, in a shaking water bath. A small aliquot of the heated suspension was plated on fresh PDA culture medium and examined periodically to evaluate fungal growth (according to
For morphological characterization, strains were cultivated on PDA (Difco, USA), Malt Extract Agar (
The phylogenetic analysis was performed using the aligned sequences of the concatenated three-gene dataset with 1301 characters (627 for LSU, 204 for
Bayesian 50% majority rule consensus tree based on an LSU/
Preliminary physiological analysis comprised all the isolates obtained in this study (data not shown). However, as no significant statistical difference was observed among the isolates under the different tested conditions, the final analysis consisted only of data regarding a copy of the culture DSM 106916. No growth was observed for the fungus after exposure to the heat tolerance protocol and therefore, it was classified as non-heat tolerant and non-heat activated. Results for NaCl tolerance test are shown in Suppl. material
Asexual morph: mycelium consisting of septate, smooth hyphae, gradually becoming widen, thick-walled, darker and developing into meristematic chains of conidia. Conidia dark brown, thick-walled, smooth, rugose, globose with single central septa resulting from the differentiation of toruloid-like hyphal cells. Sexual morph: unknown.
Members of
Asexual morph: mycelium consisting of septate, smooth hyphae, gradually becoming widen, thick-walled, darker and developing into meristematic chains of conidia. Conidia dark brown, thick-walled, smooth, rugose, globose with single central septa resulting from the differentiation of toruloid-like hyphal cells. Chlamydospores not observed in culture. Sexual morph: unknown.
Named after the old Latin name of Coimbra (
Portugal, Coimbra (
In memory of our late colleague Ludgero Avelar.
Phylogenetic analysis based on the concatenated ITS rDNA, LSU and
Mycelium initially consisting of branched, septate, smooth, subhyaline to pale green, 2–3 μm wide hyphae. Hyphae moniliform, gradually becoming widen, thick-walled, darker and developing into meristematic conidial chains. Conidiophores micronematous. Arthroconidia dark brown, thick-walled, smooth, sometimes rugose, globose, measuring 3.5–6 × 4.5–6 μm, single central septa, resulting from the differentiation of intercalary or terminal toruloid-like hyphal cells. Sexual morph unknown.
On 6 weeks old PDA plates, colonies growing slowly, to 8 mm in diameter, cerebriform, irregular, raised centrally, often moist, deeply immersed into agar, pale pink, with scarce velvety, pale pink-hyaline, aerial, short hyphae and well-defined, small, white, and glossy margin; pale pink on reverse. After at least 2 months, colonies become fully mature and melanized, olivaceous brown-black on top and on reverse. On 6 weeks old
Portugal.
Portugal, Coimbra (
Here we describe
The clustering of the environmental sequences obtained by
The limestones used in the construction of the Old Cathedral of Coimbra hail from unique areas of Portugal (namely Ançã and Portunhos, near Coimbra), and similar stone structures were exported and used on several “Our Ladies of the O” statues and in the portal of the Royal Hospital in Santiago de Compostela (Spain). We hypothesize that the transportation of limestone to such places might have contributed to the dispersion of this organism and, thus, explain the detection of
Physiological tests allowed us to characterize the retrieved isolates as non-heat tolerant, non-heat activated, xerophilic, halotolerant (enduring NaCl concentrations up to 20%), and facultative alkaliphiles. Ecologically, these traits are somewhat more similar to those of
Although
Regarding stone monuments exposed to the environment, microcolonial black fungi are one of the main culprits of stone biodeterioration and are responsible for severe aesthetic, biochemical, and biophysical alterations (
We are grateful to the Direcção Regional de Cultura do Centro (DRCC), the staff and technicians from the Old Cathedral of Coimbra (Sé Velha) and the University of Coimbra for their kind collaborations. We also thank Miguel Mesquita, for kindly providing the photographs of the sampling site. This work was financed by FEDER- Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020- Operational Programme for Competitiveness and internationalization (POCI) and by Portuguese funds through Fundação para a Ciência e a Tecnologia (FCT) in the framework of the project POCI-01-0145-FEDER-PTDC/EPH-PAT/3345/2014. João Trovão was supported by Programa Operacional Capital Humano (POCH; co-funding by the European Social Fund and national funding by MCTES), through a “FCT- Fundação para a Ciência e Tecnologia” PhD research grant (SFRH/BD/132523/2017). Fabiana Soares was supported by POCH (co-funding by the European Social Fund and national funding by MCTES), through a “FCT- Fundação para a Ciência e Tecnologia” PhD research grant (SFRH/BD/139720/2018). Nuno Mesquita was supported by POCH (co-funding by the European Social Fund and national funding by MCTES), with a Post-Doc Research grant (SFRH/BPD/112830/2015). Catarina Coelho was supported by Portuguese funds through “FCT – Fundação para a Ciência e a Tecnologia” in project IN0756 - INV.EXPLORATORIA - IF/01061/2014. Igor Tiago acknowledges an Investigator contract reference IF/01061/2014.
Figure S1. Sampling site
species data
a) Cloister of the Old Cathedral of Coimbra; b) lateral view of the Santa Maria Chapel; c) Particular art-piece from where the studied fungi were retrieved (photos by Miguel Mesquita)
Table S1. Fungal strains used in the phylogenetic analysis
phylogenetic data
Figure S2. Growth of
statistical data
Figure S3. Growth of
statistical data
Figure S4. Representative drawing of
species data
a) initial simple, branched, septate hyphae becoming toruloid-like and strongly melanized; b) differentiated, toruloid-like hyphae with chains of arthroconidia; c) and d) arthroconidia (scale 10 μm).