Preliminary Studies on Fungal Contamination of Two Rupestrian Churches from Matera (Southern Italy)

: The Sassi, a UNESCO World Heritage Site and its rupestrian churches, are richly decorated and visited by thousands of visitors every year. It is important to preserve this heritage which shows signs of deterioration due to abiotic and / or biotic factors. Aiming to carry out in the future an environmental-friendly restoration, a screening of the fungi present on walls and frescoes of two rupestrian churches “ Santa Lucia alle Malve ” and “ La Madonna dei derelitti ” located, respectively, in the “Sasso Caveoso” and in the “Sasso Barisano” was performed. Isolation and characterization of fungal species from investigated sites was carried out. Total genomic DNA (gDNA) was extracted from pure fungal cultures and subsequently utilized in PCRs using primers that amplify a portion of the ribosomal DNA (ITS5 / ITS4) or the β -tubulin gene (Bt2a / Bt2b). The amplicons were directly sequenced. Obtained nucleotide sequences were compared to those present in the GenBank (NCBI) showing a very high similarity (99–100%) with the following species: Parengyodontium album , Alternaria alternata , Cladosporium cladosporioides , Lecanicillium psalliotae , Meyerozyma guilliermondii and Botryotrichum atrogriseum . All sequences from this study were deposited in the EMBL database. Detailed knowledge about fungi isolated from stone is indispensable not only to counter / reduce the structural and aesthetic damage but also to protect the health of both guardians and visitors who may develop di ﬀ erent pathologies due to the spores di ﬀ used in the environment.


Introduction
A great number of Historical Cultural Heritage assets selected and protected by UNESCO is composed of monuments made of stone or other materials and among these edifices, the rupestrian churches are the oldest ones.One of the UNESCO World Heritage Sites (WHS) containing rupestrian churches is the Sassi and the Park of the Rupestrian Churches of Matera, inscribed in 1993 situated in the southern part of Italy, within the town of Matera (Basilicata Region, Italy).This WHS is one of the most superb, integral examples of troglodyte settlement found in the Mediterranean region harmoniously inserted into the natural landscape and ecosystem.All rupestrian churches located there can be considered without any doubt as historical treasures being richly decorated and visited by thousands of visitors every year, testifying the human presence from very old times and the strong desire to transmit to the next generations traditions and culture.In this view, the safeguarding of the cultural heritage located in Matera, The European Capital of Culture in 2019, is of primary importance not only for the cultural aspects but also for the economic ones.
Amongst the historical and cultural assets of Sassi and the Park of the Rupestrian Churches of Matera, two churches named "Santa Lucia alle Malve" and "La Madonna dei derelitti" also known as "della Scordata" are also included.The first church, located in the "Sasso Caveoso" site, was the first female monastery of the Benedictine Order from the VIII century.It is one of the most important churches of Matera providing access to only one part to the public.This church is beautifully decorated and holds a few antique frescoes."La Madonna dei derelitti" is situated in the "Sasso Barisano" site.It is a very simple and poorly structured architectural monument with its internal walls decorated with frescoes.However, at present only one fresco, named Saint Nicholas, is still visible.Both churches were excavated into stone blocks mainly composed of calcite, in approximately the same period (Late Middle Ages).
Unfortunately, these precious historical and cultural possessions of humanity, which can provide scientific information and emotional connection, had been exposed over the years to a heavy degradation process.This was caused by abiotic and/or biotic factors (biodeterioration) that depreciated stone surfaces and frescoes [1].The negative actions of these factors can often be observed as aesthetic changes (discoloration) and structural damages (defects and cracks).In addition, harmful effects of the microbial growth such as biofilm formation, biomineralization, degradation of organic binders are also very common [2][3][4][5][6][7][8].
The presence of microorganisms including fungi on the walls and frescoes, particularly inside closed areas, influenced the air quality and could also be linked to several human health problems such as asthma and other respiratory illnesses especially in people with immune system deficiencies, [9][10][11][12].
The planning and application of prevention and restoration strategies to protect the cultural/historical assets is impossible without a complete understanding of the entire microbial diversity present.Therefore, the identification of microorganisms is compulsory being the first step within this complex process [13][14][15][16][17][18].The cultivation and subsequent identification of the microbes colonizing the cultural/historical monuments which will offer some knowledge for the biodeterioration nevertheless, need to be further supported by other functional tests assessing the microbiological effects on decay.
Although, studies on microbial community such as fungi and bacteria responsible for deterioration of stone temples or churches were already undertaken [19][20][21] investigations on the edifices from The Sassi and the Park of the Rupestrian Churches of Matera are still very few or absent.A recent study reported by Caneva et al. [22] on the Crypt of the Original Sin (Matera) dealt with changes in biodeterioration patterns (BPs) on mural paintings.Microscopy and biomolecular methods aid to finally obtain effective restoration strategies.However, to our knowledge, studies on biodeterioration of the two rupestrian churches "Santa Lucia alle Malve" and "La Madonna dei derelitti" have not been reported so far.
The objective of this study was to perform an initial screening of the fungal species present on the interior walls and frescoes of the two above mentioned rupestrian churches with the final aim to accomplish, in the near future, their environmental-friendly restoration and also to evaluate the possible effects of fungi on human health.

Materials and Sampling
Two rupestrian churches,"Santa Lucia alle Malve" and "La Madonna dei derelitti", showing advanced signs of deterioration such as discoloration, cracks, fissures and exfoliation (Figure 1a-d) were investigated to eventually identify fungi present (Figure 1).
A non-destructive sampling from interior walls was carried out using a small cotton sterile swab soaked in sterile distilled water.The sampling was gently applied once on the surface of four different points (S1-S4) for each rupestrian church.
The cotton swabs were immediately placed into sterile vials containing 1 mL of double distilled water.Then, they were brought to the laboratory and stored in the fridge at 4 • C until used.A non-destructive sampling from interior walls was carried out using a small cotton sterile swab soaked in sterile distilled water.The sampling was gently applied once on the surface of four different points (S1-S4) for each rupestrian church.
The cotton swabs were immediately placed into sterile vials containing 1 mL of double distilled water.Then, they were brought to the laboratory and stored in the fridge at 4°C until used.

Fungi Isolation and Morphological Analyses
In order to isolate the cultivable fungal species, from each sample, 100 µL of suspension was directly plated on Petri dishes with Potato Dextrose Agar (PDA) amended with kanamycin (1 mg/L) and streptomycin (1 mg/L).The plates were placed in an incubator at 24 ± 1 °C in dark and incubated for 7 days.The pure fungal cultures obtained were used for further morphological and molecular analyses.Preliminary identification of pure fungal isolates was carried out using a microscope (Axioscope, Zeiss-Germany) according to the macroscopic and microscopic features of the isolates.

Genomic DNA Isolation
Mycelium was scraped from the surface of the pure fungal colony and finely ground using liquid nitrogen.The genomic DNA (gDNA) was extracted from approximately 100 mg of each sample using  c,d) of the two rupestrian churches "Santa Lucia alle Malve" and "La Madonna dei derelitti".Photos from the interior (c,d) of the two rupestrian churches also show the S1-1, S1-2, S1-3, S1-4, S2-1, S2-2, S2-3 and S2-4 sites from where non-invasive sampling was performed.To investigate overall fungal species present, material from four sampling points of each site was combined into two samples corresponding to (A) "Santa Lucia alle Malve" church and (B) "La Madonna dei dereliti" church.

Fungi Isolation and Morphological Analyses
In order to isolate the cultivable fungal species, from each sample, 100 µL of suspension was directly plated on Petri dishes with Potato Dextrose Agar (PDA) amended with kanamycin (1 mg/L) and streptomycin (1 mg/L).The plates were placed in an incubator at 24 ± 1 • C in dark and incubated for 7 days.The pure fungal cultures obtained were used for further morphological and molecular analyses.Preliminary identification of pure fungal isolates was carried out using a microscope (Axioscope, Zeiss-Germany) according to the macroscopic and microscopic features of the isolates.

Genomic DNA Isolation
Mycelium was scraped from the surface of the pure fungal colony and finely ground using liquid nitrogen.The genomic DNA (gDNA) was extracted from approximately 100 mg of each sample using the NucleoSpin Plant II™ (Macherey-Nagel, Germany) kit following manufacturer's instructions.
The quantity and quality of the recovered gDNAs was checked by readings at Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) at 260 nm and 260/230 nm wavelength.The gDNA was stored at −20 • C until further analyses.

PCR Amplification, Sequencing and Sequences Analyses
The gDNA of each pure fungal isolate was subjected to Polymerase Chain Reaction (PCR) using oligos ITS5 and ITS4 [23] or primers Bt2a and Bt2b [24] which amplified a fragment of the Internal Transcribed Spacer (ITS) and the beta tubulin gene (TUB-2), respectively.For PCR reaction, 4-6 µL (100 ng) of each gDNA extracted was employed as template in 25 µL mixture using 1 unit of Phire Hot Start II DNA Polymerase Enzyme (Thermo Scientific Inc., Wilmington, USA).The cycling protocol used for ITS amplification was the following: an initial denaturation at 98 • C for 5 min (1 cycle); followed by denaturation at 98 • C for 5 s; annealing at 58 • C for 5 s; extension at 72 • C for 20 s (40 cycles) and a final extension at 72 • C for 1 min (1 cycle).In case of β-tubulin gene the cycling protocol was the same as for ITS only with a slight modification of the annealing temperature which was set at at 62 • C. All PCR reactions were carried out using a Bio-Rad T100™ Thermal Cycler (Hercules, California, USA).PCR products were detected by electrophoresis in 1.2% (w/v) agarose gels, pre-stained with SYBR Safe DNA Gel Stain (Invitrogen Inc., Carlsbad, CA, USA) and photographed.
The amplicons were sequenced by BMR Genomics (Padua, Italy), using Illumina technology and the same primers as for the PCR.All nucleotide sequences obtained in this study were compared to those already existing in the GenBank (NCBI) public database for similar genes/regions using the Basic Local Alignment Search Tool (BLASTn) program [25].In order to correctly choose the identified taxa available in public database, nucleotide sequences of fungi from uncultured/unidentified samples and those with questionable identification were excluded.

Phylogenetic Investigations
The ITS region nucleotide sequences from this study along with other ITS sequences of the same species downloaded from the GenBank (NCBI) were used in phylogenetic investigations.Only three/four nucleotide sequences/species were carefully chosen with a very high identity (99-100%).
Nucleotide sequences obtained in this study along with those retrieved from the GenBank database (Table 1) were aligned by ClustalW multiple sequence alignment program (http://www.ebi.ac.uk/ clustalw) [26][27][28] followed by manual correction for same length adjustment.The phylogenetic analyses were performed in the MEGA-X phylogeny package [29].Note: * The β-tubulin (TUB2) partial gene was amplified only for this fungal species.** This fungal species was used as an outgroup only for the phylogenetic analysis involving B. atrogriseum isolates.a The year refers to the publication year found in the NCBI GenBank database for the respective nucleotide sequence.
In case of the phylogenetic analysis of ITS region, the final dataset had 50 nucleotide sequences with 403 positions.All sites were treated equally for the analysis and gaps were treated as missing data.Codon positions included were 1st + 2nd + 3rd + Noncoding.All positions containing gaps and missing data were eliminated (complete deletion option).The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model [30].In order to test the phylogenetic tree robustness statistical verification by bootstrap analysis (1000 replicates) was performed [31].
The β-tubulin sequences from this study along with three similar sequences downloaded from the NCBI database, were processed in a separate phylogenetic analysis similarly to the ITS [29][30][31] with the only difference that the final nucleotide sequences alignment had seven nucleotide sequences and there were a total of 401 positions in the final dataset.The nucleotide sequence (Acc.no.KP900708) of Subramaniula thielavioides (Arx, Mukerji and N. Singh), strain CBS 122.78, a closer species from the Chaetomiaceae, was chosen as an outgroup.

Fungi Isolation and Morphological Characterization
A total number of 36 pure fungal culture isolates were obtained on PDA media from the two rupestrian churches in Matera as shown in Table 2.The observation of morphological features by light microscopy allowed the identification of six fungal genera (Table 2).

Molecular Identification of the Fungal Species
The PCR performed with the ITS primers produced a single amplicon of 531 to 589 bp in size.Instead, PCR employing Bt primers, yielded a unique amplicon of 452 bp (Table 2).
The ITS sequences, from this study, after comparison with those present in the GenBank (NCBI), showed a 99-100% sequence identity with the ITS sequences belonging to the following species: The β-tubulin nucleotide sequences from this study had a 98.99% sequence identity with the same gene belonging to Botryotrichum atrogriseum J.F.H. Beyma species (accession numbers are presented in Table 1).
DNA sequence information for all fungal isolates generated from the present study was deposited in the European Nucleotide Archives (ENA; https://www.ebi.ac.uk/ena) of the EMBL database (Table 2).

Phylogenetic Identification of the Fungal Species
Phylogenetic trees based on ITS nucleotide sequence data constructed with both distance and maximum parsimony (MP) using the Mega-X phylogeny package gave identical results.Thus, only the phylogenetic tree based on Maximum Likelihood (ML) analysis will be presented.
In particular, the unrooted ML tree showing the phylogenetic position of the ITS rRNA gene sequences, retrieved from the internal walls and frescoes of the two rupestrian churches in Matera, in relation to their closest type species is shown in Figure 2.
same gene belonging to Botryotrichum atrogriseum J.F.H. Beyma species (accession numbers are presented in Table 1).
DNA sequence information for all fungal isolates generated from the present study was deposited in the European Nucleotide Archives (ENA; https://www.ebi.ac.uk/ena) of the EMBL database (Table 2).

Phylogenetic Identification of the Fungal Species
Phylogenetic trees based on ITS nucleotide sequence data constructed with both distance and maximum parsimony (MP) using the Mega-X phylogeny package gave identical results.Thus, only the phylogenetic tree based on Maximum Likelihood (ML) analysis will be presented.
In particular, the unrooted ML tree showing the phylogenetic position of the ITS rRNA gene sequences, retrieved from the internal walls and frescoes of the two rupestrian churches in Matera, in relation to their closest type species is shown in Figure 2. All identified isolates of a given genus closely affiliated (bootstrap value of 100%) to the species already present in the GenBank database (Table 1) are simultaneously well separated from all other analyzed genera (Figure 2).Phylogenetic analysis based on β-tubulin gene, demonstrated that the three isolates of B. atrogriseum from this study are closely related to the same species already present in the GenBank databasegrouping all in the same clade (Figure 3).In addition, the three B. atrogriseum isolates, originated from the rupestrian churches of Matera, clustered in the same subclade with an excellent bootstrap value of 99%.They were also clustering with one of the B. atrogriseum CCF-5752 downloaded from the GenBank with relatively high (90%) bootstrap support.
values are reported as a percentage of 1000 replications).Bootstrap values > 50% were indicated at the nodes.The scale bar indicates the number of expected changes/site.
All identified isolates of a given genus closely affiliated (bootstrap value of 100%) to the species already present in the GenBank database (Table 1) are simultaneously well separated from all other analyzed genera (Figure 2).
Phylogenetic analysis based on β-tubulin gene, demonstrated that the three isolates of B. atrogriseum from this study are closely related to the same species already present in the GenBank databasegrouping all in the same clade (Figure 3).In addition, the three B. atrogriseum isolates, originated from the rupestrian churches of Matera, clustered in the same subclade with an excellent bootstrap value of 99%.They were also clustering with one of the B. atrogriseum CCF-5752 downloaded from the GenBank with relatively high (90%) bootstrap support.

Discussion
It is well known that the abiotic (wind, water, ice, thermal oscillations etc.) and biotic factors (e.g., fungi, bacteria, algae, lichens) can cause structural and esthetic damage to the historical monuments matrix exhibited as degradation, deterioration and decay phenomena [32][33][34].
More precisely, fungi are frequently associated to biodeterioration of stone monuments [35][36][37].They are responsible for various modifications of the substrate observed as discolorations, exfoliations, loss of material, bioweathering, surface deposits, etc. due to acid corrosion, enzymatic degradation and mechanical attack [33,35,36].Several genera, like Alternaria, Cladosporium, Verticillium, Penicillium, Engyondontium are commonly linked to biodeterioration of cultural and historic heritage [6,[38][39][40][41].Therefore, identifying the main features of these fungi, assess their nutritional needs and collect information related to their growth and development will help to select future specific protocols to be applied in order to contrast any present degradation process or prevent a forthcoming one.

Discussion
It is well known that the abiotic (wind, water, ice, thermal oscillations etc.) and biotic factors (e.g., fungi, bacteria, algae, lichens) can cause structural and esthetic damage to the historical monuments matrix exhibited as degradation, deterioration and decay phenomena [32][33][34].
More precisely, fungi are frequently associated to biodeterioration of stone monuments [35][36][37].They are responsible for various modifications of the substrate observed as discolorations, exfoliations, loss of material, bioweathering, surface deposits, etc. due to acid corrosion, enzymatic degradation and mechanical attack [33,35,36].Several genera, like Alternaria, Cladosporium, Verticillium, Penicillium, Engyondontium are commonly linked to biodeterioration of cultural and historic heritage [6,[38][39][40][41].Therefore, identifying the main features of these fungi, assess their nutritional needs and collect information related to their growth and development will help to select future specific protocols to be applied in order to contrast any present degradation process or prevent a forthcoming one.
Furthermore, indoor mycoflora can have negative effects on human health causing respiratory illnesses, keratosis, and pulmonary diseases especially in immune-suppressed patients [42,43].The presence of fungal spores in elevated concentrations inside of edifices increases the risk to human health.One of the most often fungal genera linked with allergic asthma and allergic rhinitis are Alternaria and Cladosporium [44,45] which were both found in our study.
A. alternata is a very common fungal species widely present on different substrates and environments [9,34,35].This species was extensively found in cultural and historical heritage sites associated with substrate alterations like discolorations [6,14,46,47].Furthermore, A. alternata spores are recognized as being very common powerful aeroallergens [48,49].Usually intense exposure to A. alternata allergens happens in the open-air.However, this fungus is very often found in indoor environments where the level of exposure to aeroallergen increases.In addition, human exposure to 9 of 13 fungal spores of A. alternata has strongly been associated with increased asthma severity [44,50,51].Thus, the presence of A. alternata may be considered as a potential threat for the health of guardians and visitors in cultural heritage sites.
Previous studies by Gutarowska et al. [18], who reported the presence of P. album from cultural heritage assets, are in agreement with the outcomes from this study.Fungi from genus Engyondontium (Parengyondontium) are linked to biodegradation phenomena [18,52] observed on different matrices and also connected to human health like fungaemia in immunocompromised patients [53] endocarditis [54], brain abscess [55] or keratitis [56].
C. cladosporioides, another fungal species isolated from the two investigated sites in Matera, is a common microbial agent on stone monuments and often associated with the accelerated degradation process of ancient wall painting discoloration [4][5][6]18].Exposure of humans to Cladosporium spores is also reported to be linked to allergic reactions [57].
Results from this study, reporting the presence of the L. psalliotae species are in accordance with the research of Sareela et al. [4] who already described L. aranerarum (another species of genus Lecanicillium) from marble surface, frescoes and biofilm of Roman catacombs of St. Domitilla and St. Callistus in Rome.
M. guilliermondii was also detected from both walls and frescoes in this study.To our knowledge, there are no reported studies on M. guilliermondii from cultural heritage monuments.Maciel et al. [58] revealed the M. guilliermondii involvement in human candidose disease.The same fungus was reported by Corte and co-workers to be present on food and other environmental niches [59].Its diversity was investigated and also some clues for its speciation were provided.Furthermore, very recently De Marco et al. [60] investigated the genomes of another Meyerozyma species and presented new and significant data regarding the M. guilliermondii species-complex.
The recent work by Wang et al. [61] revealed few different species of Botryotrichum B. piluliferum (originated from walls), B. murorum (isolated from ceiling tiles) and B. peruvianum (isolated from walls) while B. atrogriserum examined in their study originated even from corn field soil, dung of rabbit or mountains but not from walls.
The results from this study report the presence of B. atrogriseum on interior walls and frescoes from the two rupestrian churches in Matera.
Moreover, some fungal species identified in this study, A. alternata, C. cladosporioides and P. album, were previously associated with walls [4,18,41,52].To our knowledge, there are no reported studies on M. guilliermondii, B. atrogriseum and L. psalliotae from cultural heritage monuments.However, a close relative of L. psalliotae and B. atrogriseum namely L. aranerarum and B. piluliferum, and B. peruvianum were showed to originate from wall samples [61].
The Chaetomium genus, that B. atrogriseum also belongs to, is known to produce potentially harmful metabolites which may have an effect on the health of the visitors and curators, therefore its investigation is of great importance [62,63].
In order to avoid problems for human health, due to spores spread [64], it would be appropriate to improve the air quality inside heritage sites through the use of air-filters or by controlling fungal presence.Besides, a seasonal variation of fungal populations may occur over time and consequently the risks for human health can vary [65,66].
The control of fungi in cultural heritage assets is an important issue which was investigated using various ways to limit their development and growth such as the use of natural substances employed as green conservation strategies and the utilization of engineered nanomaterials [67][68][69].
Results from this preliminary study contributed to furnish new and useful knowledge about the presence, morphology and molecular characterization of fungi found on the walls and frescoes of two rupestrian churches of Matera.Considering the information obtained here about these fungi, along with future programmed culture-independent studies and investigations concerning their implication in the degradation of the two cultural heritage sites and their effects on the health of guardians and visitors, it will be possible to start a concrete planned and preventive conservation

Sustainability 2020 , 13 Figure 1 .
Figure 1.General view of the environmental context (a,b) and interior (c,d) of the two rupestrian churches "Santa Lucia alle Malve" and "La Madonna dei derelitti".Photos from the interior (c,d) of the two rupestrian churches also show the S1-1, S1-2, S1-3, S1-4, S2-1, S2-2, S2-3 and S2-4 sites from where non-invasive sampling was performed.To investigate overall fungal species present, material from four sampling points of each site was combined into two samples corresponding to (A) "Santa Lucia alle Malve" church and (B) "La Madonna dei dereliti" church.

Figure 1 .
Figure 1.General view of the environmental context (a,b) and interior (c,d) of the two rupestrian churches "Santa Lucia alle Malve" and "La Madonna dei derelitti".Photos from the interior (c,d) of the two rupestrian churches also show the S1-1, S1-2, S1-3, S1-4, S2-1, S2-2, S2-3 and S2-4 sites from where non-invasive sampling was performed.To investigate overall fungal species present, material from four sampling points of each site was combined into two samples corresponding to (A) "Santa Lucia alle Malve" church and (B) "La Madonna dei dereliti" church.

Figure 2 .
Figure 2. Phylogenetic tree based on Maximum Likelihood method and Kimura 2-parameter model, analyzed for partial Internal Transcribed Spacer (ITS) sequence (total of 403 positions in the final dataset) of samples from two rupestrian churches in Matera identified in this study and closely related sequences downloaded from the NCBI database.The tree with the highest log likelihood (−2108.93) is shown.Statistical evaluation of the tree topology was performed by bootstrap analysis (bootstrap values are reported as a percentage of 1000 replications).Bootstrap values > 50% were indicated at the nodes.The scale bar indicates the number of expected changes/site.

Figure 3 .
Figure 3. Phylogenetic tree based on Maximum Likelihood method and Kimura 2-parameter model, analyzed for partial β-tubulin sequence (total of 401 positions in the final dataset) of samples from two rupestrian churches in Matera identified in this study and closely related sequences downloaded from the NCBI database.The tree with the highest log likelihood (−926.30) is shown.Statistical evaluation of the tree topology was performed by bootstrap analysis (bootstrap values are reported as percentage of 1000 replications).Bootstrap values >70% were indicated at the nodes.The scale bar indicates the number of expected changes/site.

Figure 3 .
Figure 3. Phylogenetic tree based on Maximum Likelihood method and Kimura 2-parameter model, analyzed for partial β-tubulin sequence (total of 401 positions in the final dataset) of samples from two rupestrian churches in Matera identified in this study and closely related sequences downloaded from the NCBI database.The tree with the highest log likelihood (−926.30) is shown.Statistical evaluation of the tree topology was performed by bootstrap analysis (bootstrap values are reported as percentage of 1000 replications).Bootstrap values >70% were indicated at the nodes.The scale bar indicates the number of expected changes/site.

Table 1 .
List of the species, isolates/strains and their GenBank accession number downloaded from the NCBI database and used in the phylogenetic analysis.

Table 2 .
Fungi recovered from the two rupestrian churches with their phylogenetic resemblance and GenBank accession numbers.
a Number showing the length of the PCR products (base pairs) obtained in this study, sequenced and further analyzed.