**6. Clinical effects of SGLT2 inhibitors in diabetes mellitus**

Currently, there are seven SGLT2 inhibitors approved for clinical use. They are given orally and absorbed by the intestine. Due to higher selectivity for SGLT2 versus SGLT1, inhibition of intestinal SGLT1 can be avoided, though it is still possible at high oral doses. At pharmacological doses, their serum levels achieved are too low to inhibit SGLT1 in other organs (**Table 2**).

Clinical effects observed for different SGLT2-I will be described and discussed together for the sake of clarity.

### **6.1 Effects on diabetes and metabolism**

In T2DM, upregulation of SGLT2 expression increases its Tm by around 20%. SGLT2-I is filtered in glomeruli and inhibits glucose reabsorption in S1 segment of proximal tubule leading to a reduction of 30–50% in Tm of SGLT2 [67].

In the presence of functional SGLT2, less than 10% of glucose is absorbed through SGLT1; therefore, it is expected that SGLT2 inhibitor therapy would lead to around 90% reduction in Tm but the observed decrease of only 30–50% in Tm can be explained by higher amount of SGLT1-mediated glucose reabsorption [70].

#### **Table 2.**

*Preparation strength and SGLT2 versus SGLT1 selectivity of various approved SGLT2 inhibitors.*

During preclinical studies with animal models of diabetes as well as in clinical studies with both T2DM and T1DM patients, it has been demonstrated that prolonged SGLT2-I therapy decreased fasting and prandial plasma glucose levels, reduced HbA1C, and improved oral glucose tolerance. They also exerted nephroprotective effects, reduced blood pressure, and increased utilization of fatty acid substrates; thus, they also conferred metabolic benefits [68–73].

SGLT2-I have insulin-independent mechanism of action. They can be used both as monotherapy as well as in combination with other OAHs [74, 75]. Clinical trials have shown that SGLT2-I are effective when administered in combination with metformin [74, 76–78], metformin plus sulfonylurea [79], insulin [80], DPP4 inhibitors [77], and thiazolidinediones [75].

SGLT2-I do not increase the risk of hypoglycemia like other OAH agents. The filtered renal glucose load is directly correlated with plasma glucose level, and there is compensatory increase in SGLT1-mediated glucose reabsorption when SGLT2 is blocked [81]. The other mechanism preventing hypoglycemia is the glucagon secretion from pancreatic alpha cells due to SGLT2 inhibition [31].

SGLT2-I therapy in patients with T2DM increases both plasma glucagon and endogenous glucose production. Despite such physiological changes, patients on SGLT2-I have lower plasma glucose levels than those receiving placebo, possibly because of increased glycosuria and improved insulin sensitivity [82, 83].

SGLT2-I administration changes body metabolism and shifts it to enhanced usage of fat for metabolic needs; consequently beta-hydroxybutyrate levels in plasma increase [69]. The metabolic inflexibility characteristically seen in patients with T2DM and nondiabetic insulin-resistant subjects is an inability to switch from predominantly fatty acid oxidation during fasting state to predominantly glucose oxidation in fed state [84]. Lack of variability in measured respiratory quotient (RQ ) between fasting and fed states has been observed as an evidence of metabolic inflexibility. SGLT2-I reduce whole body fasting RQ which is indicative of increased oxidation of fatty acids and amino acids, suggesting its partial restoration [85].

As a consequence of improved glucose homoeostasis, SGLT2-I may slow down glucotoxicity-mediated degeneration of beta cells and thus may also slow down the progression of T2DM. Experimental diabetic animal models on SGLT2-I have shown an improvement in both beta cell mass and functions [86]. Improvement in insulin sensitivity is another beneficial aspect [82, 83].

#### **6.2 Effects on blood pressure**

Increased urinary excretion of glucose and sodium leads to mild diuresis coupled with urinary sodium loss. Reduction of extracellular volume occurs which has a favorable effect on blood pressure, and the magnitude of the effect is most apparent in patients with preexisting hypertension [87].

Empagliflozin causes a reduction in systolic blood pressure (SBP) of 4–6 mmHg, whereas diastolic blood pressure (DBP) was similar to placebo with no increase in the heart rate [88]. Similarly, canagliflozin at 300 mg/day resulted in a reduction in SBP of 5.1 mmHg [89].

In analysis of 12 studies with dapagliflozin at 10 mg/day, reduction of 4.4 mmHg and 0.5 mmHg in SBP and DBP, respectively, was seen with no increase in the heart rate compared to placebo [90].

#### **6.3 Effects on body weight**

On SGLT2-I therapy, initial weight loss could result from osmotic diuresis. However, sustained weight loss over the period is a consequence of renal glucose

**85**

*SGLT2 Inhibitors Therapy in Type 2 Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.84152*

**6.4 Effects on plasma lipids and plasma uric acid**

cholesterol (HDL) has been observed [76, 78, 94].

*6.5.1 Effects on cardiovascular disease*

all plausible protective mechanisms.

carcinoma and may result in liver cirrhosis also.

required no dose adjustments [98, 99].

*6.5.2 Effects on liver disease*

and high CVD risk [96].

trial [91].

excretion leading to a caloric deficit of about 280 calories/day. This translates into

SGLT2-I therapy has been associated with alteration in serum lipid profile. Small increase in low-density lipoprotein cholesterol (LDL) and high-density lipoprotein

Any increase in serum cholesterol is considered to be a risk factor for the development of heart failure and CVDs. However, it may not be relevant clinically, as empagliflozin has been shown to have a protective effect on MACE in EMPA-REG

They also promote uric acid excretion and reduce uric acid level in the blood [94] which may contribute to their protective effect on the development of diabetic

In EMPA-REG trial, empagliflozin has shown a lower risk for MACE as well as

In CANVAS Program, canagliflozin has been found to reduce the risk of cardiovascular death or hospitalization for heart failure in patient with a history of T2DM

Lately, DECLARE-TIMI 58 trial demonstrated the CV safety of dapagliflozin in patients with T2DM who had or were at risk of CVDs. It was found to reduce hospitalization for heart failure as has been seen with other SGLT2-I [97].

Both empagliflozin and canagliflozin has got FDA approval for cardiovascular risk reduction in T2DM patients who are at high risk for such events. The mechanism of cardiovascular protection exerted by SGLT2-I is unknown and seems to be glucose independent. Apart from improved glycemic control, the pleiotropic effects of SGLT2 inhibitors which include their ability to lower blood pressure, reduction in intraglomerular pressure and albuminuria, and amelioration of volume overload are

T2DM affects metabolism in the liver and may manifest as hepatic steatosis and nonalcoholic steatohepatitis (NASH). NASH increases the risk of hepatocellular

In preclinical studies on rodent models, SGLT2-I have been found to ameliorate nonalcoholic fatty liver disease (NAFLD) and NASH [70–72]. Although SGLT2-I are metabolized by the liver, studies involving patient with mild or moderate

hepatic impairment showed dapagliflozin and empagliflozin were well tolerated and

Canagliflozin has shown improvement in serum aminotransferases and gammaglutamyl transferase levels in patients with T2DM [100]. In humans, ipragliflozin has shown reduction in liver fat in patients with T2DM and NAFLD [101]. Empagliflozin addition to the standard treatment of T2DM and NAFLD signifi-

cantly reduced liver fat, and improved ALT levels were seen [102].

nephropathy observed in the EMPA-REG renal study [73, 80, 95].

for cardiovascular death and has been found to be protective [88].

decrease in body weight by 1–3 kg along with decreased visceral adiposity. In the EMPA-REG trial, empagliflozin resulted in weight loss of about 2 kg [91]. The average weight loss achieved with dapagliflozin and canagliflozin stands

between 1 and 3%, while other studies report a loss more than 5% [92, 93].

#### *SGLT2 Inhibitors Therapy in Type 2 Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.84152*

*Type 2 Diabetes - From Pathophysiology to Modern Management*

[77], and thiazolidinediones [75].

substrates; thus, they also conferred metabolic benefits [68–73].

tion from pancreatic alpha cells due to SGLT2 inhibition [31].

insulin sensitivity is another beneficial aspect [82, 83].

in patients with preexisting hypertension [87].

**6.2 Effects on blood pressure**

SBP of 5.1 mmHg [89].

rate compared to placebo [90].

**6.3 Effects on body weight**

During preclinical studies with animal models of diabetes as well as in clinical studies with both T2DM and T1DM patients, it has been demonstrated that prolonged SGLT2-I therapy decreased fasting and prandial plasma glucose levels, reduced HbA1C, and improved oral glucose tolerance. They also exerted nephroprotective effects, reduced blood pressure, and increased utilization of fatty acid

SGLT2-I have insulin-independent mechanism of action. They can be used both as monotherapy as well as in combination with other OAHs [74, 75]. Clinical trials have shown that SGLT2-I are effective when administered in combination with metformin [74, 76–78], metformin plus sulfonylurea [79], insulin [80], DPP4 inhibitors

SGLT2-I do not increase the risk of hypoglycemia like other OAH agents. The filtered renal glucose load is directly correlated with plasma glucose level, and there is compensatory increase in SGLT1-mediated glucose reabsorption when SGLT2 is blocked [81]. The other mechanism preventing hypoglycemia is the glucagon secre-

SGLT2-I therapy in patients with T2DM increases both plasma glucagon and endogenous glucose production. Despite such physiological changes, patients on SGLT2-I have lower plasma glucose levels than those receiving placebo, possibly because of increased glycosuria and improved insulin sensitivity [82, 83].

SGLT2-I administration changes body metabolism and shifts it to enhanced usage of fat for metabolic needs; consequently beta-hydroxybutyrate levels in plasma increase [69]. The metabolic inflexibility characteristically seen in patients with T2DM and nondiabetic insulin-resistant subjects is an inability to switch from predominantly fatty acid oxidation during fasting state to predominantly glucose oxidation in fed state [84]. Lack of variability in measured respiratory quotient (RQ ) between fasting and fed states has been observed as an evidence of metabolic inflexibility. SGLT2-I reduce whole body fasting RQ which is indicative of increased oxidation of fatty acids and amino acids, suggesting its partial restoration [85]. As a consequence of improved glucose homoeostasis, SGLT2-I may slow down glucotoxicity-mediated degeneration of beta cells and thus may also slow down the progression of T2DM. Experimental diabetic animal models on SGLT2-I have shown an improvement in both beta cell mass and functions [86]. Improvement in

Increased urinary excretion of glucose and sodium leads to mild diuresis coupled

Empagliflozin causes a reduction in systolic blood pressure (SBP) of 4–6 mmHg, whereas diastolic blood pressure (DBP) was similar to placebo with no increase in the heart rate [88]. Similarly, canagliflozin at 300 mg/day resulted in a reduction in

In analysis of 12 studies with dapagliflozin at 10 mg/day, reduction of 4.4 mmHg and 0.5 mmHg in SBP and DBP, respectively, was seen with no increase in the heart

On SGLT2-I therapy, initial weight loss could result from osmotic diuresis. However, sustained weight loss over the period is a consequence of renal glucose

with urinary sodium loss. Reduction of extracellular volume occurs which has a favorable effect on blood pressure, and the magnitude of the effect is most apparent

**84**

excretion leading to a caloric deficit of about 280 calories/day. This translates into decrease in body weight by 1–3 kg along with decreased visceral adiposity.

In the EMPA-REG trial, empagliflozin resulted in weight loss of about 2 kg [91]. The average weight loss achieved with dapagliflozin and canagliflozin stands between 1 and 3%, while other studies report a loss more than 5% [92, 93].

### **6.4 Effects on plasma lipids and plasma uric acid**

SGLT2-I therapy has been associated with alteration in serum lipid profile. Small increase in low-density lipoprotein cholesterol (LDL) and high-density lipoprotein cholesterol (HDL) has been observed [76, 78, 94].

Any increase in serum cholesterol is considered to be a risk factor for the development of heart failure and CVDs. However, it may not be relevant clinically, as empagliflozin has been shown to have a protective effect on MACE in EMPA-REG trial [91].

They also promote uric acid excretion and reduce uric acid level in the blood [94] which may contribute to their protective effect on the development of diabetic nephropathy observed in the EMPA-REG renal study [73, 80, 95].


**2. State of the art**

There can be found several inventions to assist in the installation of asphalt coating, most of them based on one or more torches. In 1993, the patent ES 2041192 – entitled "Method and apparatus for applying a bituminous sheet to a substrate" [6] relates a preheated constant temperature roller (close to 300°C), wherein bituminous sheets slide toward their installation (**Figure 1**).

The patent DE-A 1652399-Device for heating and gluing a rolled material on a substrate [7] was published in 1971 (**Figure 2**). This patent claimed for a mechanism that allows the installation of a sheet by the application of heat to it. It includes a series of torches placed along all the bituminous sheet width. It has some disadvantages, for example, the direction of operator walking is backward, and this may cause falls from a certain dangerous height. Also, it prevents the operator from seeing what is happening behind his back, which could cause injury to other operators or apply the sheet on material remains causing fissures. Also, the heat application on the sheet is done from a great distance with respect to the installation point, and the bitumen primer that must be put on the substrate where the sheet is installed does not heat up. Moreover, the linear heating of the bituminous sheet causes that heat must be applied for a longer time, since it heats up from room temperature. Besides, there is only one general gas-regulating valve, which causes a perceptible pressure drop between the first torch and the last one. Last, the device could be too long, making it difficult to transport and place it on the construction site.

The patent US 4725328 [8]—a single ply roofing applicator, from 1988, presents the same characteristics commented for the previous mechanism, differing only in

**Figure 1.** *ES 2041192 patent design [6].*

**Figure 2.** *DE-A 1652399 patent design [7].*

two aspects. First, two torches are incorporated, placed perpendicular to the substrate, which are placed at both ends to promote adherence in the overlaps. Secondly, two rollers are added to each side of the mechanism to improve adherence to the overlaps by means of pressure against the substrate. It also shares some disadvantages with the previous mechanism, such as backward direction of operator walking, the linear heating of the bituminous sheet, and that there is only one gas-regulating valve.

In 1982, the patent US 4354893 [9]—combination roofing material unrolling and heat applying apparatus, was published. This patent describes a lighter design than the described above, since it dispenses with the roller system and the base to support the gas cylinder. It incorporates an extra handle, which permits the torches to get toward the roll as it unwinds, therefore enhancing heat application. As the previous ones, the direction of operator travel is backward, the warming is done from room temperature, that there is only one gas-regulating valve and, in addition, the unrolling system of the bituminous sheet is carried out by means of a bar incorporated into the axis of the roll. This is not efficient because the bituminous sheet is flexible and tends to deform. The appearance of folds that can cause problems for water filtration is favored.

Other significant patent is US2006037710 [10]—a membrane applicator. It varies in two aspects with respect to the previous patents: a gas cylinder is supported by a base,

*New Trends in Industrial Equipment for the Improvement of Asphalt Roofing Process DOI: http://dx.doi.org/10.5772/intechopen.101795*

and it includes the incorporation of a roller on one flank of the mechanism to guarantee the adherence of the overlap. Their downsides, again, are the reverse direction of travel, but also heat must be applied for a longer time since it heats up from room temperature, and to have only one gas-regulating valve, which is also a pitfall.

As well as patents, it is interesting to analyze what commercialized mechanisms exist. Among them, Seal-Master 1030 (Schäfer Technic GmbH) [11] is very relevant. It is a machine that allows the continuous installation of bituminous sheets in civil engineering works. It is mainly characterized by the large roller on which the bituminous sheet roll is placed for unwinding. This mechanism is not suitable for use on roofs due to its great weight and the use of sheets of greater length than those marketed for waterproofing roofs. It also incorporates a base to place the gas cylinder that is used to supply the torches that are located in the lower part of the mechanism. Finally, the roller used to ensure adherence of the bituminous sheet throughout its width also stands out. On the Schäfer Technic GmbH website, it is indicated that the weight of the set is 660 kg, which makes this mechanism not easy to transport. A site crane would be needed to raise it to the roof, and overload problems on punctual spots of the roof could seriously damage the structure of the construction.

Unify-ER (RES Automatisation Contrôle) [12] is characterized by its remote control (controlled by an operator) and by a greater number of mobile mechanisms than the rest of the systems, which makes this mechanism considerably increase in weight and makes it difficult to use on roofs. It also has a frame on which to place the

**Table 1.**

*Details of the characteristics to be improved in the different patents and commercialized products.*

gas cylinder, a system of rollers to improve the unrolling of the bituminous sheet, and some side stops that guide the roll and prevent folds from appearing when the sheet is placed on the substrate. It is based on the patent US 8887782—membrane applying apparatus, from 2014. The company claims for an installation speed of 1 m/ min, which means a rate of 18 rolls per hour employing two workers. This leads to an installation of 480 m2 a day per person.

The principal drawbacks of these two systems are that they are too large to operate on small roofs, too heavy, and challenging to transport. In addition, they have an electronic and battery system, which increases the possibility of having failures that prevent the continued use of the device.

On the other hand, the devices FORSTHOFF-P2 (Forsthoff GmbH) [13] and BITUMAT B2 (Leister Technologies AG) [14] can be found. They only serve to heat the sheets in the overlaps, which is an essential requirement for a good waterproofing of the roofs, but they do not heat the rest of the sheet.

And finally, there is Bitumenbrenner (Bamert Spenglerei GmbH) [15], which allows the entire surface of the bituminous sheet to be heated as it uses four individualized torches equally spaced between them, spreading across the width of the sheet. This system also needs the heat to be applied for a longer time, since it heats up from room temperature, at least two operators are required (one of them unrolls the sheet while the other applies heat to it), and it does not allow a correct overlap between sheets, this being the most important process in the installation of waterproofing.

**Table 1** presents main characteristics of previous state-of-the-art semiautomatic roofing systems. **Table 1** includes those features that should be improved or changed.


**1.1 Aim and objectives**

The aim of this chapter is to introduce an algorithm that combines the two approaches so as to achieve the design of robust networks and test it on a number of instances that are representative of communication network problems. On the one hand, the network must be highly reliable from a probabilistic point of view (its reliability) assuming that the probabilities of failure of all links and sites are known. On the other hand, the network structure must be topologically robust; for this, node or edge connectivity levels between pairs of distinguished nodes are required. This means that between all pairs of distinguished nodes there exists a given number of edge paths or disjoint nodes. Then, once a minimum threshold for reliability (e.g., 0.98) is set, the algorithm here introduced:

Performed research indicates that literature pertaining to algorithms on design topologies which consider both approaches (survivable networks and network reliability) is scarce. The works on the design of robust networks in general fix a level of node/edge global connectivity of the network and try to design a network at the lowest possible cost that satisfies that level (e.g., 2-node-connectivity) [1]. Nevertheless, there are contexts where this combination of approaches is imperative and demanded. For example, in the context of military telecommunication networks, it is required that the networks are topologically very robust (e.g., 3-nodeconnectivity) and at the same time that they are extremely reliable from the network reliability approach's point of view, surpassing very high reliability levels. Another example of the application of the combined model is the logical distribution of highly dangerous merchandise on a country's roads. In such a context, two things are desirable: high reliability in the connection of points of distribution (i.e., "reliable" roads) and high levels of connectivity between the points that must exchange cargo (availability of alternative roads to possible road cuts, traffic saturation, etc.).


An extensive search was made among peer reviewed literature, proceedings to conferences, and books. The literature was identified using international databases, such as Scopus (https://www.scopus.com, 29 December 2021), Science Direct (https://www. sciencedirect.com, 29 December 2021), Web of Science (http://www.webofknowledge.com, 29 December 2021) and Google Scholar (https://scholar.google.com, 29 December 2021), that were consulted by using keywords such as 'wall paintings', 'mural paintings', 'frescoes', 'fungi', 'biodeterioration', 'microbial deterioration', and 'biodeteriogenic agents'. The thematic databases of ICCROM (International Centre for the Study of the Preservation and Restoration of Cultural Property) and the Italian ISCR (Istituto Superiore per la Conservazione e il Restauro) were also consulted, being important reference institutions in the field. Such sources were fundamental in the search of literature related to congresses and reports, that are not found by the most common scientific reference tools. The search covered more than 50 years, dating back to the first papers published in the 1960s (Figure 1), even if mostly of the papers containing useful taxonomic information were published after the 1980s.

**Figure 1.** PRISMA flow diagram of the search process.

#### *2.2. The Database*

A list of all the entries retrieved, corresponding to taxa identified at both species and genus level, was compiled. Few were the entries referring to taxa above the rank of genus and they are listed at the end of the database. Current names of the taxa were reported according to the Index Fungorum (www.indexfungorum.org, 20 January 2022). The synonyms under which the different species were eventually reported in the analyzed papers were also indicated. Those entries recorded on paintings that have been the subject of multiple studies over the years, such as Takamatsuzuka and Kitora tumuli in Japan [23–25], were reported once accompanied by all bibliographic references.

#### 2.2.1. The Geographic Localization of the Study Sites

The geographical locations and coordinates of the studied monuments were retrieved using Google Maps. The graphical representation on a map of sites distribution was performed using the 3D Map function of the Excel package. Some papers dealt with an unidentified number of monuments, as in the case of 'Monasteries in Romania' [21], 'several churches' in Northern Portugal [26], or 'Ajanta caves' in India [27–32]. In these cases, one or more sites were counted, depending on the details provided by the authors within the studied area and on their geographical distribution. Instead, some papers dealt with different monuments from the same area (e.g., different buildings in the historical site of Herculanum, Italy), that have been considered as a single site for the purpose of this study. This is why the total number of monuments is higher than the total number of geographical sites assessed in the statistical analyses.

#### 2.2.2. Isolation and Identification Methods Used

The following information has been recorded: the type of culture media used, the growth temperature and incubation time, and the methods used for isolates identification.

#### 2.2.3. The Types of Environments

The environments that housed the wall paintings object of the studies considered were grouped into five categories, based on whether they were hypogean or not, as well as the expected ventilation, confinement, and moisture conditions. The following are the definitions of the categories:

but sheltered from rain and ventilation; they all experience seasonal and daily relative humidity and temperature fluctuations.

5. **O-SPE**: Open and Semi-Protected or Protected Environment; they include buildings' walls or collapsed caves, which are among the most exposed sites to biodeterioration risks.

#### *2.3. Statistical Analyses*

A data dissimilarity matrix was inferred using the Jaccard's dissimilarity index [33], and a hierarchical cluster analysis was performed on this matrix using the UPGMA method. Two dendrograms relating dissimilarities between sites and entries were plotted. Entries identified at the genus level were considered as diverse elements. The Silhouette index was used to resolve the optimal number of clusters [34]. A contingency table between the obtained clusters and the environmental categories to which they belonged was also constructed to assess the relationship between their data. An indicator species analysis of the individual clusters was performed, which identifies associations between entries or combinations of entries and clusters, using the Indval index [35,36].

All analyses were performed with the R Software with the packages ade4, vegan, gclus, cluster, vegclust, and indicspecies.

#### **3. Results**

#### *3.1. The Fungal Data Set*

A total of 103 papers dealing with the fungal deterioration of wall paintings were collected, regarding 107 different monuments grouped in 74 sites. A total of 378 fungal entries were obtained, consisting of 1209 records belonging to 173 genera and 260 species (Table 1).

**Table 1.** List of the fungal entries retrieved from the different papers grouped by genera, in association with the corresponding references and the environmental categories where they have been registered.

The taxonomic distribution of the total fungal diversity and within different types of environments, at phylum and order level, is reported in Figure 2. Ascomycota was the dominant phylum, ranging from 89 to 97% (except for the environment O-SPE where a great proportion of undetermined taxa was retrieved) and accounting for 100% of the fungal entries in 40 out of 74 sites. The other two phyla identified were Basidiomycota (1–6%), more abundant in C-NHE, and Mucoromycota, reaching a maximum value (2.35%) in C-NHE (Figure 2A).

**Figure 2.** Taxonomic composition of the fungal diversity recorded on wall paintings on the total dataset and in relation to the different environments, at (**A**) phylum and (**B**) order level.

The entries were distributed in 39 orders. Eurotiales and Hypocreales were the most represented ones, together accounting for more than half of total fungal diversity. Pleosporales, Capnodiales, Sordariales, and Chaetothyriales were also well represented orders within Ascomycota. Basidiomycota accounted for a maximum of around 6% of all taxa, with Agaricales, Helotiales, and Saccharomycetales being the most abundant ones accordingly. Mucorales was the most abundant order within the phylum Mucoromycota.

The accuracy in taxa determination varied among the different papers analyzed, with many entries identified only at genus level. In fact, a total of 107 out of 378 entries (28.3%) referred to genera not determined at the species level, and it was not possible to quantify the number of possible different species belonging to these genera within different papers. A total of 61 genera (35.2%) were represented by a single species. *Aspergillus* and *Penicillium* (order Eurotiales) were the most frequently recorded genera and were represented by a greater number of species, 40 and 46, respectively, present in 44 (59.4%) and 32 (43.2%) sites, which increased to 54 (73%) and 51 (68.9%) when those sites where the genera were reported as undetermined at the species level were also considered. Their contribution to the total number of records was significant, with 219 (18.1%) and 154 (12.7%) records, respectively. Other genera frequently recorded were *Alternaria* and *Fusarium* with seven species each; *Acremonium*, *Cladosporium,* and *Trichoderma* with six species; *Curvularia* and *Talaromyces* with five species; and *Chaetomium* with four species.

#### *3.2. The Geographic Distribution of the Study Sites*

The data came from 107 monuments, grouped in 74 sites and distributed among 19 different countries. The countries where they were reported as well as the different number of sites were graphically represented in Figure 3.

#### *3.3. Isolation and Identification Methods*

A dominance of culture-based methods associated with morphological identification (58%) and target regions sequencing (31%) was recorded, the latter has become progressively dominant since the 2000s. The culture media used varied depending on the research purposes and included Czapek Dox agar (CZ), malt extract agar (MEA), malt agar (MA), potato dextrose agar (PDA), Sabouraud dextrose agar (SAB), and oatmeal agar (OA) among the most frequently used. The use of mycological agar (MYC), Cook's Rose Bengal (CRB), and yeast peptone dextrose (YPD) was sporadic. Data on growth temperature and incubation time were frequently missing, accounting for 31.25% (Figure 4A, green) and 54.12% (Figure 4B, green) of all papers, respectively. This trend was particularly evident in the

earliest papers where more attention was paid to fungal species than to the conditions used to isolate them (e.g., [40,47,84,88]). The most frequent temperature settings were 25 ◦C (35%) and within the range 27–32 ◦C (21.25%). When reported, the incubation frequently corresponded to 7 days (31.76%) (Figure 4B). When isolated strains were identified by molecular approaches, the identification was performed targeting different barcoding regions, such as the complete internal transcribed spacer (ITS), a part of it (ITS1), portions of 18S (SSU), 26S (LSU), and β-tubulin.

**Figure 3.** Geographic distribution of the reviewed study's 107 monuments. Nations are indicated with the international alpha-3 code: ITA: Italy, EGY: Egypt, THA: Thailand, PRT: Portugal, CHN: China, ROU: Romania, AUT: Austria, GER: Germany, JAP: Japan, IND: India, RUS: Russian Federation, ESP: Spain, SRB: Republic of Serbia, CUB: Cuba, FRA: France, KOR: Republic of Korea, SVK: Slovak Republic, CHE: Swiss, GBR: Great Britain. In the blue rectangle Cuba.

**Figure 4.** Values/ranges of (**A**) growth temperature and (**B**) incubation time recorded in culture-based protocols.

#### *3.4. Distribution of Fungal Entries in Different Environments*

The distribution of the sites among the environmental categories revealed a predominance of wall painting recorded in C-NHE, which alone accounted for 54% of the sites. The hypogean environments accounted for 36.5% of the sites, distributed between C-HE (18.9%) and NC-HE (17.6%). The remaining two categories, SC-NHE and O-SPE, were less represented, comprising 4.1% and 5.4% of the sites, respectively.

The cluster analysis at the level of different sites resulted in a general dispersion, with no distinct clusters retrieved. Several clusters consisted of one or few sites highly different one to each other (13-11-14-3-15-10-9-7-12). Other clusters (4-5-6) were slightly more similar to each other and contained many entries (Figure 5).

**Figure 5.** Cluster dendrogram. Cluster analysis on the Jaccard distance of the composition at the level of the different sites. Sites belonged to the following categories: Site1–Site14 C-HE; Site15–Site27 NC-HE; Site 28–Site67 C-NHE; Site 68–Site70 SC-NHE; Site 71–Site74 O-SPE. The references associated to each site are reported in Supplementary Table S1.

The contingency table highlighted that the obtained clusters did not have a strong correlation with the suggested environmental categories (Table 2). However, some clusters showed some affinities: cluster 15 with cat. 4 (SC-NHE) (aff. index 17); cluster 6 with cat. 5 (O-SPE) (aff. index 12.8); clusters 2,4,5 with cat. 3 (C-NHE) (aff. indexes 8.1, 9, and 6.2, respectively); cluster 4 with cat. 2 (NC-HE) (aff. index 5.9); cluster 1 with cat. 1 (C-HE) (aff. index 8.2).

**Table 2.** Contingency table and affinity indices. (**A**) Contingency table between the obtained clusters and the environmental categories to which they are correlated and (**B**) their relative affinity indices.

When the correlation among different entries was analyzed, very scattered results were retrieved (Figure S1). However, some entries or combinations of them showed some affinities (specificity and fidelity above 0.5) with certain clusters such as *Alternaria alternata* with cluster 2 (Indval Index = 0.75); *Acrothecium* sp. and *Penicillium* sp. with cluster 4 (Indval Index = 0.70 and 0.66, respectively); *Parengyodontium album* and *Rhinocladiella*-like with cluster 5 (Indval Index = 0.70 and 0. 64); *Actinomucor elegans*, *Penicillium pancosmium*, *Acremoniella atra*, *Alternaria angustiovoidea*, *Scolecobasidium anomalum*, *Scolecobasidium lascauxensis*, when considered in combination, the entries *Trichoderma* sp. and *Verticillium* sp. with cluster 1 (Indval Index = 0.70, 0.70, 0.70, 0.70, 0.70, and 0.70, respectively); and *Akanthomyces lecanii* with cluster 9 (Indval Index = 0.93).

#### **4. Discussion**

#### *4.1. The Fungal Data Set*

Even if fungi have been suggested as secondary colonizers of painted mural substrates, they are among the most common microbial life-forms present in these environments and the primary cause of their biodeterioration [1,13,14]. The wide biodiversity observed confirms the potential key role of fungi in such colonization process and suggests a combination of causes that can favor their growth. Their broad enzymatic activities allow them to grow on every type of material, or wherever they find organic matter. Fungi recorded belonged to species generally reported from natural environments like soils, plants, and air where they live as saprotrophs, as well as plant and animal parasites and pathogens. A detailed survey of their diversity and distribution should become a prerequisite before any restoration measures in order to prevent further damages [39]. Most records belonged to Ascomycota, with Eurotiales being the most common order, due to the prevalence of *Aspergillus* and *Penicillium* genera. The former was one of the most frequently isolated genera, with *A. flavus* and *A. niger* among the more frequently recorded species. As reported in the literature, even from the first older papers in this field, these two genera, along with *Alternaria*, *Fusarium*, *Cladosporium*, *Mortierella*, *Chaetomium*, and *Acremonium*, are among the most common deteriogens of such paintings [1,11,15,31,39,46,62,78,87,91]. These taxa are ubiquitous, and their frequent occurrence is due to the production of numerous conidia, which are widespread in the environment because they are easily dispersed by air. A diversity of filamentous fungi, with the most predominant genera *Penicillium*, *Cladosporium*, *Aspergillus*, and *Trichoderma*, were also isolated from mural paintings of the Parish Church of Santo Aleixo (Portugal). Their dehydrogenase activity was determined, as an indicator of the presence of metabolic active cells to allow a deeper insight on the deteriogenic role of the isolates [19].

Species of these genera were recorded on indoor frescoes in numerous monasteries in Romania, possibly favored by the organic components and vegetal pigments used, as well as high moisture levels caused by frequent rainwater penetration, which also resulted in the formation of efflorescences [55]. *Cladosporium* species can cope in a variety of harsh environmental conditions thanks to their low nutritional requirements (i.e., in oligotrophic conditions). Otherwise, *Chaetomium* species are proteolytic and cellulolytic ascomycetes, favored by nutrient-rich substrates [22,65,117]. They were reported as the most frequent microfungi on the frescoes of the St. Damian Monastery in Assisi (Italy) [46] and on frescoes in a Serbian church [22]. Furthermore, a community of *Aspergillus*, *Penicillium*, *Cladosporium*, and *Chaetomium* species was recorded from Medieval wall paintings in Styria (Austria), forming spots of different colors [39]. This group of genera was dominant on two deteriorating frescos in St Clare's Refectory of the Monastery of St Damian in Assisi [46].

Hypocreales was the second most abundant order, accounting for 18% of total fungal diversity, within which *Acremonium*, *Trichoderma*, and *Fusarium* were among the most common genera. Hypocreales is one of the largest orders of filamentous ascomycetes and exhibits a broad range of ecologies, ranging from plant-associated nutritional modes to animal pathogens (e.g., insect pathogens) and mycoparasites [118]. *Neocosmospora solani*, recorded in Thailand, India, Japan, and France; *Simplicillium lamellicola*, recorded in Russia; and *Clonostachys rosea* [48,58], recorded in Japan, are examples of mycoparasitic species, while *Parengiodontium album* is an insect parasite and was recorded in several countries (Germany, Russia, Romania, Austria, Italy, and England) [109]. The recurrent presence of mites and insects pointed out their possible role in spreading fungi on painted surfaces [46,119].

Finally, the plant pathogen species *Fusarium oxysporum* has been shown to produce an extracellular pinkish pigment that disfigures and aesthetically damages colonized mural paintings and stone surfaces with permanent stains [78].

Phylum Basidiomycota was present with several occasional species, mostly represented by one or two records, and comprises litter, soil, and wood-saprotrophs, ectomycorrhizal, epiphyte, and plant-pathogen species. Their occurrence must be regarded as sporadic, potentially aided by root penetration. The possible role of roots as a carrier for rhizosphere microorganisms, like a dripping line for water condensation, and as an organic carbon source by root exudates has been hypothesized [57,120]. In any case, a Basidiomycete was also recorded at the entrance of Roman catacombs [121], possibly due to spores carried by water infiltrations and germinating using organic nutrients from the soil and/or the phototrophic biofilm.

Mucoromycota was present with few species and records, and black meristematic fungi were rarely recorded as well. These latter may grow on a wide range of substrates and are resistant to a variety of environmental stresses, as well as being widely distributed epi- and endolithically on monuments [122,123]. Although the biodiversity of black fungi on historical monuments is not fully elucidated, recent samplings indicate that they are also present on wall paintings and that their rare finding could be linked to the isolation protocols used, generally favoring fast-growing species [124]. Two new species of the genus *Neodevrisia* have been found in the restricted sampling area of the Vallerano cave and another, still undescribed, from Maijishan grottoes [45,99,108]. *Scolecobasidium lascauxensis* and *S. anomalus* were isolated and described from black stains in Lascaux Cave, France [85,115], while the chaetothyrialean black fungi *Cladophialophora*, *Exophiala*, and *Phialophora* have been reported from different sites [23,24,42,98].

Yeasts have been rarely reported, such as Saccharomycetales (Ascomycota) that usually grow by individual yeast cells or *Rhodotorula* spp. (Basidiomycota) often linked to pink/orange stains due to the release of carotenoids [19,93].

Among those more commonly reported, some species such as those belonging to the genera *Alternaria*, *Fusarium*, *Aspergillus*, *Penicillium*, and *Cladosporium* may be responsible for annoying allergic and toxic reactions suffered by conservationists and visitors [81,125,126]. *Alternaria alternata* is a very common fungal species, frequently recorded on frescoes. Its spores are recognized as common powerful aeroallergens, and indoor environments offer higher levels of exposure to this risk than open-air [59,127]. Records of *Fusarium* species have also been reported, such as *F. solani* in the Lascaux caves [97] and *F. oxysporum* in many sites. They are mainly plant pathogens, but they can also be the causal agents of human mycoses [97,128,129]. Some *Aspergillus* species are pathogenic to humans and animals and are responsible for clinical manifestations (https://www.aspergillus.org.uk/species-archive/, 29 December 2021). Among these, *A. fumigatus* is a human pathogenic fungus recorded on frescoes within different environments (3,11,22,29,42,49,70,71), causing infections in humans which can be fatal in immunocompromised patients (61). *Aspergillus flavus* has been frequently recorded in monasteries, churches, temples, caves, and tombs, mostly due to their numerous aerosolized spores. It mainly grows in the soil, but it is also a facultative and opportunistic pathogen of both animals and plants, producing mycotoxins that are highly harmful to humans [130].

In light of the above, the identification of the species deteriorating wall painting is needed for the protection of restorers and visitors. However, the temperature values characterizing confined and semi-confined sites are generally too low for potential pathogenic fungi. In fact, truly thermophilic fungi which cannot grow at temperatures below 20 ◦C are not active in these environments, at least during winter in temperate regions.

#### *4.2. Geographic Distribution*

Considering the geographic distribution of the data, just one site among the studied paintings comes from the Americas (the Cathedral of Havana at Cuba) [82]. The highest number of records was from Europe, with 70 monuments, mostly representative of hypogean environments and of churches and historical buildings, with a considerable prevalence of Italian monuments (39). A total of 26 monuments were from Asia, while the 13 African ones were all from Egypt.

This distribution arises from the old tradition of people of the Euro-Mediterranean area of using such artistic expression, starting from the old prehistoric caves to the Etruscan and Greek-Roman traditions until the consolidated use both in the decoration of Christian churches and historical buildings [2]. In the case of the Egyptian area, the recorded taxa derived from the old tombs of the Pharaohs [54,72,73,77,78], and similarly in East Asia, the tradition is mainly found in kings' and Emperors' tombs [23–25,38]. Most of the ancient paintings in buildings do not exist anymore, due to frequent rebuilt or remake of the materials [131].

Our results suggest that the monuments studied were often confined to restricted geographic areas. In any case a wider geographic distribution than that recorded may be possible, as a number of sites may have escaped the search. In fact, even if formally available on the web and on the major repositories, because of the language barriers, some studies could not be taken into account.

#### *4.3. Isolation and Identification Methods*

Culture-based methods favor the growth of microorganisms best fitting with the laboratory conditions used (namely, culture media, temperatures, and incubation times). In this study, we found that the most frequent experimental settings were favorable to fastgrowing, highly-sporulating fungi, with the use of culture media rich in easily accessible carbon sources, alongside short incubation times and optimal growth temperatures favoring their sporulation. Otherwise, lower growth temperatures (≤20 ◦C), wide temperature ranges, different isolation media, and a longer incubation time could enlarge the detectable culturable fraction.

Since the early 2000s, molecular phylogenetic methods have highlighted the limitations of morphological identification, allowing us to gain a better understanding of the kingdom of Fungi [132]. Nowadays, the identification by barcode regions sequencing is a common practice. Even if the nuclear ITS region has been recognized as a fungal barcode, its discriminating power changes within the taxonomical groups, and other/more barcode regions are often necessary to have a reliable identification [133]. This is the case of the identification of species within large groups, as *Fusarium*, *Penicillium*, *Aspergillus*, and *Cladosporium* genera, where cryptic species can be detected only by sequencing multiple molecular markers [134].

In detail, *Fusarium* species determination has been best made with the combined phylogeny of protein coding genes such as elongation factor (TEF1), RNA polymerase (RPB2) and the partial β-tubulin (BT2) gene [134]. To discriminate between *Penicillium* and *Aspergillus* species, β-tubulin (BT2) and calmodulin (cmdA) genes have been proposed as secondary barcodes, respectively [135,136]. While the most phylogenetic informative markers for *Cladosporium* were TEF1 and actin gene (actA), ITS sequences being identical for species of the same complex [137,138].

The correct identification of strains is required in order to provide restorers more information about strains' ecology and degradative potential. In this light, standardized identification protocols should be implemented.

High throughput sequencing methods have recently been applied to cultural heritage purposes. These methods represent a powerful tool to define the whole fungal diversity present but not necessarily to deepen the mechanisms and the main actors of the deterioration phenomena [139]. The combination of culture-based and molecular methods should be used for a better understanding of deterioration processes. Indeed, pure cultured microorganisms represent the key to uncover settlers' physiological and ecological traits, as well as representing a resource for many in silico applications and barcoded identifications [123,124].

#### *4.4. Distribution of Taxa in the Different Environments*

The most prevalent sites were confined non-hypogean environments, which are characterized by varied thermo-hygrometric temperatures and air movement. Hypogean (both confined and non-confined), where nutrients and humidity can favor fungal growth were represented as well.

Temperature and relative humidity are among the environmental parameters most important to microbial colonization capability, and in the case of heterotrophs, a certain amount of nutrients is also needed [4,140]. It is well known that fungi rapidly grow when relative humidity is higher than 65% and when a certain quantity of nutrients is available. The low values of temperatures, even if are not favorable for microbial growth by themselves, have a positive effect in contributing to increase in humidity, favoring water condensation on surfaces. Walls, especially in hypogean environments, generally provide these requirements [1]. Temperatures in confined environments are generally more stable than in non-confined environments, where daily and seasonal changes may occur, with ranges that have effects on microbial settlement. Elevated moisture values and stable temperatures have been reported as ideally suited to promote microbial growth on surfaces in catacombs sites [7,94,141]. Indeed, the highest risk occurs when high humidity is coupled with high temperature values, and negative effects of rising temperatures arise only if their highest values can strongly influence the humidity values [142]. In the case of hypogea, the underground conditions favor the maintaining of humidity.

Air movement differences between confined, semi-confined, and non-confined environments were expected to alter the number and type of fungal species recorded as well as incoming nutrients from the outside environment. A great proportion of entries in the database belonged to soil and litter dwellers such as saprotrophs, producing numerous spores that are well adapted to air-borne dispersal, and therefore, air ventilation may have a significant impact on the risk of contamination [143]. The more limited air volume movement of confined mural paintings compared to semi-confined ones was suggested to decrease the number of air-borne dust particles, with biofilm communities relying more on internal interactions between different microorganisms than on the external organic inputs [17]. Among the first aerobiological studies, Savulescu and Ionita reported a greater number of isolates inside the studied monasteries than outside of them, probably due to a more favorable microclimate inside the church, which favors the development of microorganisms [55]. Pangallo and colleagues proposed for the first time a comparative analysis of the microbial component of paintings and the surrounding air to gather information on the origin of fungal contamination [70]. Aside from the importance of aerobiological studies for the conservation and prevention of microbial attacks on indoor painted surfaces [144], these studies have received little attention. In light of the large number of fungal species potentially harmful for restorers and visitors, constant monitoring of air spore quality and concentration, as well as the use of air filters to reduce fungal spores concentrations, would be required for site conservation [62,101,144,145].

Significant correlations between the different taxa and the various environmental categories have not been recorded. Indeed, such data is not the result of the absence of a correlation between fungal growths and environmental conditions but can be consequence of several other influencing conditions that hide it. In fact, many are the ecological requirements that shape the ecological niches of the different species (i.e., the limiting factors), but the most conditioning factors are those that result in a quantity proximal to the upper or lower tolerance limit of an organism [146]. Then, for the various sites examined, some factors may become more relevant if their values are closer to the tolerance limit of certain organism, but this does not mean that other parameters do not play a role [147].

Indeed, our results may be influenced by the wide number of taxa in the wide geographic distribution of sites and by the different methodologies used to characterize the fungal diversity. In fact, different sampling techniques and isolation conditions were used within the studies we analyzed. Other factors that allow fungi to thrive and/or survive in a variety of conditions are their wide nutritional versatility and range of adaptations. The presence of numerous genera that are widespread and highly sporulating and hence present in all the environmental categories must also be considered. The absence of evident correlations could have been determined by the absence of distinct boundaries between the categories identified, with overlapping microclimatic conditions which could have resulted in overlaps within their respective microbial communities. Finally, the heterogeneity of the data, with taxa identified at the genus or species level, may have resulted in dispersed clusters and hampered the ability to demonstrate any relationship.

This result seems to be in line with other studies. The influence of environmental factors such as temperature, relative humidity, and the opening or closure of the temples was not evident for fungal growths on wall paintings of 12 archaeological sites in the central and western parts of Thailand [76]. Furthermore, a stronger relationship with the age of five caves in China than with the environmental conditions, such as temperature and relative humidity, was proposed to explain the observed differences in fungal communities [64]. In two distinct mural paintings, instead, the differences recorded in the microbial communities were associated to the different organic input origin (i.e., wine cellar evaporation, and insect exuvia/excrements) and the microclimatic conditions. The more humid conditions favored the growth of actinomycetes, bacteria, and dark-pigmented fungi, while the other showed a biofilm, mainly dominated by xerotolerant and patchy growing sporulating fungi [17]. Differences in fungal communities were also recorded on mural paintings of two subterranean ancient Chinese tombs dating back over 1700 years, mostly due to variations in interior temperature and relative humidity as well as to their history and drawing techniques used [51].

Other significant concerns could be related to the identification of the isolated species, which was initially based solely on morphological observation. Indeed, phylogenetic molecular approaches are nowadays routinely applied, providing a universal tool for accurately identifying fungal species.

New methodologies such as omics techniques are now available; however, they rarely provide information at species or genus level, and there is no guarantee that the recorded taxa are actively growing. Moreover, culture-dependent approaches may not provide a real picture of the microbial diversity actively growing at the sampling time. This is because not all fungi actively growing on the deteriorated substrates can grow under laboratory conditions, and fungi growing under laboratory conditions may not actively grow on the sampled surfaces. Therefore, a combination of culture-based and molecular approaches may be needed to gain a clear picture of the actual biodiversity present on the painted surfaces as well as to have strains to investigate their potential degradative roles.

#### **5. Conclusions**

This study contributes to illustrate the high fungal diversity on wall paintings and raises awareness about the fungal threat on the deterioration of such artworks. Ascomycota was the most common phylum, with Eurotiales and Hypocreales as the most common orders. Statistical analyses did not enhance core communities that can be considered characteristics of different environmental categories of sites hosting wall paintings. Our results were likely due to the heterogeneity and fragmentation of the data in the databases, the dispersed geographical area considered, and the complexity of factors which can condition the biological growths. It is therefore crucial to cover the knowledge gaps through (i) international collaborations, (ii) enlarging the isolation and cultivation protocols as to easily detect also strains different from fast growing ones, and (iii) standardizing the identification protocols. Standardizing and improving the site descriptions (e.g., repeated microclimatic data) could allow for possible relations between site and their settlers and for further comparisons among different environmental conditions.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app12062988/s1, Figure S1: Cluster analysis on the Jaccard distance of the distribution of the different entries retrieved from the papers analyzed; Table S1: List of all the references associated to each site defined in the analyses.

**Author Contributions:** Conceptualization, L.Z. and G.C.; methodology, L.Z., F.C., D.I. and G.C.; data curation, L.Z., F.C., D.I. and G.C.; writing—original draft preparation, L.Z. and F.C.; writing—review and editing, L.Z., F.C., D.I. and G.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Authors wish to acknowledge Giulio Zangari (Department of Sciences, University of 'Roma Tre') for his support in some statistical elaborations. Authors wish to acknowledge Giulia Corsetti Antonini and Matt Hudson for the revision of the English language.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Review* **Black Fungi on Stone-Built Heritage: Current Knowledge and Future Outlook**

**Filomena De Leo \*, Alessia Marchetta and Clara Urzì**

Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale F. Stagno d'Alcontres, 31, 98166 Messina, Italy<EMAIL_ADDRESS>(A.M<EMAIL_ADDRESS>(C.U.) **\*** Correspondence<EMAIL_ADDRESS>
**Featured Application: This is an updated review on black fungi as main biodeteriogens of cultural heritage stone artifacts. Colonization pattern, taxonomy, and methods to eradicate their settlement are discussed here.**

**Abstract:** Black fungi are considered as one of the main group of microorganisms responsible for the biodeterioration of stone cultural heritage artifacts. In this paper, we provide a critical analysis and review of more than 30 years of studies on black fungi isolated from stone-built heritage from 1990 to date. More than 109 papers concerning the fungal biodeterioration activity of stone were analysed. The main findings were a check list of the black fungal taxa involved in the biodeterioration of stone-built heritage, with a particular reference to meristematic black fungi, the main biodeterioration pattern attributed to them, and the methods of study including the new molecular advances. A particular focus was to discuss the current approaches to control black fungi from stone-built heritage and future perspectives. Black fungi are notoriously hard to remove or mitigate, so new methods of study and of control are needed, but it is also important to combine classical methods with new approaches to improve current knowledge to implement future conservation strategies.

**Keywords:** stone cultural heritage; black fungi; MCF; biodeterioration; control

#### **1. Background**

Stonework, such as artistic sculptures, historical buildings, monuments, archaeological sites, caves, etc., are ubiquitous across the globe, being an expression of culture, religion, aesthetics, and building techniques of populations, typical of certain historical construction periods. Due to their unicity and intrinsic value, ensuring the integrity of stone-built heritage for posterity is a critical issue. The study of biodeterioration of cultural heritage is a hot topic of broad interest to the researcher's community and the implementation of safeguard measures is one of the main goals. All materials are subjected to a natural weathering, and "biodeterioration" of stones should be considered as an integral part of bio-geo-morphogenesis [1–4]. The term "biodeterioration" defines any irreversible transformation of inorganic or organic material with economic, commercial, historic, and artistic loss caused by macro- and micro-organisms [5].

"Biodeterioration" is a very complex matter and conservators should also take into account whether the observed biologically driven phenomena can even be considered positive for the artifact.

In fact, in some cases, the presence of subaerial biofilm (SABs) may have a protective effect on the surface [6]; on the other hand, SABs developed at the interface between rock surface and air is considered the main cause of biodeterioration of stone monuments [7,8].

Microbial biodeterioration of stones is often associated with the presence of a complex community formed by chemoorganotrophic microorganisms (bacteria and microfungi) and autotrophic microorganisms (such as algae and cyanobacteria and to lesser extent

**Citation:** De Leo, F.; Marchetta, A.; Urzì, C. Black Fungi on Stone-Built Heritage: Current Knowledge and Future Outlook. *Appl. Sci.* **2022**, *12*, 3969. https://doi.org/10.3390/ app12083969

Academic Editor: Cesareo Saiz-Jimenez

Received: 1 March 2022 Accepted: 11 April 2022 Published: 14 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

autotrophic bacteria) usually embedded in an extracellular matrix EPS (in which are present DNA, enzymes, pigments, lipids, proteins, etc.). Microbial cells in the EPS show a typical biofilm lifestyle that confers resistance to hostile environments and reinforces the attachment of microorganisms on the surface [6,9].

The prevalence of one or more group of microorganisms depends on numerous factors which include the intrinsic characteristics of the material (such as lithotype, porosity, roughness, and state of preservation) that affect its "bioreceptivity" *sensu* Guillitte [10]. The species composition can vary greatly depending on climatic and microclimatic conditions such as temperature, solar irradiation, shining, nutrient and water availability, and, last but not least, the characteristics of species involved [6]. However, microbial colonization is a very dynamic process in time and space, that is the result of the interactions between microbial species and substrates. It varies continuously during the year following the seasons, and it is also under the influence of the dispersion ability of propagules in the air [11–13].

In recent years, much knowledge has been gained about rock-inhabiting black fungi, and important issues concerning their taxonomy, physiology, phylogeny, and weathering processes [14] have been clarified. However, the majority of studies concerned black fungi from natural environments [4,7,15].

In the field of cultural heritage, most reviews had as a topic the biodeterioration of stone caused by fungi in general [6,16,17]; some have focused on the microbial and fungal deterioration of various type of substrata (both organic and inorganic such as textile, parchment, wood, paper, metals, and stone) used for artworks [18]; few concerned exclusively black fungi as a cause of biodeterioration of stone monuments [19,20].

This paper aims to give an overview on the present knowledge of rock-inhabiting black fungi in the field of stone cultural heritage with reference to their taxonomy, biodeterioration pattern, methods of study, and control, with a look to a future perspectives.

A bibliographic search was carried out using such databases as Scopus (https:// www.scopus.com accessed on 17 March 2022), Science Direct (https://www.sciencedirect. com accessed on 17 March 2022), Web of Science (http://www.webofknowledge.com accessed on 17 March 2022), and Google Scholar (https://scholar.google.com accessed on 17 March 2022), that were consulted by using keywords such as 'black fungi', 'meristematic fungi', 'stone monuments', 'stone artworks', 'stone biodeterioration', 'biodeteriogenic fungi', 'fungal treatment', and 'fungal control'.

The search produced about 500 papers of which 109 were included in this paper. The updates of fungal nomenclature were searched in the databases Index Fungorum (http: //www.indexfungorum.org accessed on 22 March 2022), National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov accessed on 24 February 2022) and in the recent literature [14,21].

Nucleotide sequences were retrieved from GenBank database that is accessible from NCBI platforms (http://www.ncbi.nlm.nih.gov accessed on 14 February 2022). Molecular Evolution Genetic Analyses (Mega 11 Software) free downloadable via the URL. http: //www.megastoftware.net accessed on 14 February 2022 was employed for alignments and phylogenetic tree constructions.

#### **2. Black Fungi and Stone Monuments: An Intimate Connection**

Beginning in the 1990s, black fungi were described as one of the most likely groups of microorganisms responsible for the biodeterioration of the stone monuments [22–25] and it was confirmed in the following decades [6,18,26,27].

The term "black fungi" refers to a very huge group of dematiaceous fungi, unrelated phylogenetically, which have in common the presence of melanin in the cell wall that confers an olive brown appearance to the colony [28]. Another common characteristic is the ability to withstand hostile environments such as scarcity of nutrients, high solar irradiation, scarcity of water, high osmolarity, and low pH [15,19,29].

As reported by Gueidan et al. [30] the ancestors of black fungi were well adapted to live in oligotrophic environments such as rock surfaces or sub-surfaces, and currently they can also grow in anthropogenic habitats such as glass, silicon, organic surfaces, metals [31], or consolidants applied on the stone [9].

Their resilience is related to the extremotolerant or even polyextremotolerant characteristics of the species. The stress-tolerance is due to different factors such as: pigmentation, and in particular melanins production; mycosporine-like substances; morphological and metabolic versatility; meristematic development; and oligotrophy [32–34]. All these characteristics make them very suitable for colonizing outdoor rocks and built stones due to the fact that those surfaces can be exposed to extreme environments [17–19].

This group of fungi includes (a) fast growing hyphomycetes of epiphytic origin, recognizable under microscope by the presence of typical conidiophores and spores; (b) pleomorphic hyphomycetes that include the "black yeasts", showing a yeast-like form, and the so-called "black meristematic fungi" with a *Torula*-like growth pattern (Figure 1).

(**a**) (**b**)

**Figure 1.** Main morphological characteristic traits of MicroColonial Fungi, MCF. Dark black colonies due to the melanin production as seen (**a**) for the unidentified strain MC 655 on different cultural media after 1 month of incubation. (**b**–**d**) characteristic meristematic pattern of growth described also as *Torula*-like hyphae observed under Light Microscope. Bar is 10 μm.

Hyphomycetes and black yeasts are ubiquitous and widespread all over the world in very different habitats (e.g., soil, fresh water, sea, plants, animals, and humans) [28,35], while the meristematic black fungi, mostly isolated from stone or natural rocks, can be considered the true stone-inhabiting fungi [19,20,36,37].

In the literature, many of black fungi are reported as RIF (rock inhabiting fungi) to emphasize that the "rock" is their preferred or exclusive habitat. However, this terminology does not include their main features such as melanin production, pleomorphism, or meristematic development; for this reason, we do not use it in this context.

In the frame of cultural heritage the acronym MCF (MicroColonial Fungi) as first employed by Staley [38] is widely used for their description. It refers to the typical black cauliform-like colonies visible on the rocks and stones.

Humidity may affect the settlement of MCF on the stone artifacts as unique inhabitants or as associated with other stone colonizers. In fact, in lower or sheltered parts near the ground, where there is a sufficient availability of water, MCF are strictly associated with phototrophic microorganisms with whom, however, they do not establish a symbiotic relationship (Figure 2); in harsh, dry micro-environmental conditions, MCF become the unique colonizers [39–41].

**Figure 2.** Close association between MCF and phototrophic microorganisms. (**a**) Association seen directly on a marble sample. Magnification 400X and (**b**) after growth in the isolation medium PDA: *Chlorella*-like alga and *Coniosporium apollinis* MC 728. Magnification 80X.

Black fungi are currently classified in the Phylum of Ascomycota in the Class of Dothideomycetes and Eurotiomycetes, mainly in the order of Capnodiales, Dothideales, Chaetothyriales, Pleosporales and Cladosporiales, and Mycocaliciales [14,20,21,42].

In Table 1 are listed the genera of black fungi identified through molecular analyses that from 1997 up to date have been related to the biodeterioration of stone monuments.

*Appl. Sci.* **2022**, *12*, 3969

**Table 1.** *Cont.*

In manuscripts published prior to 1999, black meristematic fungal species that were identified without molecular analyses, such as *Hormonema dematioides*, *Lichenothelia* sp. and *Hortaea werneckii*, *Trimmatostroma* sp., are listed among the most abundant fungal species present in arid and semiarid environments in association with biodeterioration of stone monuments [3].

The molecular analyses introduced at the end of the 20th century considerably increased the knowledge about the taxonomy of the black fungi isolated from stone monuments and allowed the description of twenty-six new species and three new genera.

The new species and genera described are listed below:

*Sarcinomyces petricola* Wollenzien and de Hoog [73]; *S. sideticae* Sert and Sterflinger [70]; *Coniosporium apollinis* Sterflinger, *C. perforans* Sterflinger [43]; *C. uncinatum* De Leo, Urzì and de Hoog [44]; *C. sumbulii* Sert and Sterflinger [47]; *Phaeococcomyces chersonesos* Bogomolova and Minter [74]; *Pseudotaeniolina globosa* De Leo, Urzì and de Hoog [54]; *Capnobotryella antaliensis* Sert and Sterflinger [50]; *C. erdogani* Sert and Sterflinger; *C. kiziroglui* Sert and Sterflinger [51]; *Ochroconis lascauxensis* Nováková and Martin-Sanchez; *O. anomala* Nováková and Martin-Sanchez [69]; *Knufia marmoricola* Onofri and Zucconi, *K. vaticanii* Zucconi and Onofri; *K. karalitana* Isola and Onofri; *K. mediterranea* Selbmann and Zucconi [37]; *K. calcarecola* Su, Sun and Xiang [72]; *Exophiala bonarie* Isola and Zucconi; *Vermiconia calcicola* de Hoog and Onofri [37]; *Devriesia simplex* Selbmann and Zucconi; *D. modesta* Isola and Zucconi [55]; and *D. sardiniae* Isola and de Hoog [37].

Three new genera and 4 species were also introduced as new: *Saxophila tyrrenica* Selbmann and de Hoog, *Lithophila guttulata* Selbmann and Isola [37], *L. catenulata* Su, Sun and Xiang [72], and *Aeminium ludgeri* Trovão, Tiago and Portugal [57].

Over the years, some of the above mentioned genera and species were reclassified: in particular, *Sarcinomyces petricola* and *Phaecoccomyces chersonesos* resulted identical, and they were reclassified as *Knufia petricola* [75,76]; *Coniosporium perforans* is now a synonym with *Knufia perforans* [76]; *Devriesia* species and *Vermiconia* species were included, respectively, in the new genera of *Neodevriesia* [77] and *Vermiconidia* [21]. Hao et al. [78] proposed a revision of the genus *Ochroconis* that was established as synonymous with the sister genus of *Scolecobasidium.* However, this taxonomic accommodation has been refused by Samerpitak et al. [79,80] on the basis of phylogenetic analyses and because the old generic name *Scolecobasidium* is considered of doubtful identity for the ambiguity of type specimens; therefore, the genus *Ochroconis* that is also characterized by oligotrophism and mesophilia was maintained.

However, many questions regarding the taxonomy and phylogeny of black fungi are still unresolved and further studies are required, especially to clarify the taxonomical position and phylogeny of many species of *incertae sedis* and of strains that are preserved in the mycological collections and are not yet identified (Figure 3, Table 2).

**Figure 3.** Phylogenetic tree (Neighbour-joining, Kimura two-parameters) showing the genetic divergence among ITS rDNA sequences of meristematic black fungi retrieved from GenBank database (https://www.ncbi.nlm.nih.gov/nucleotide accessed on 14 February 2022) and listed in Table 2.

**Table 2.** ITS rDNA sequences of representative MCF isolated from stone monuments aligned in Figure 3.

**Table 2.** *Cont.*

#### **3. Mechanisms Involved in the Stone Biodeterioration**

Being well adapted to the stone habitat and being oligotrophic, this group of fungi can often act as pioneer colonizer of the stone. In fact, for their growth it is sufficient to have just a little input of nutrient coming from the surrounding environment (e.g., animal and plant particles, air pollutants, guano droppings, etc.) [81,82]. Marble exposed to different environments and laboratory experiments demonstrated that black fungi such as *Aureobasidium pullulans* can be the first colonizer of freshly exposed marble surfaces in outdoor conditions [68,83].

The presence of a source of organic matter, such as the proximity of plants and trees, can considerably increase the chances of colonization by these fungi and the consequent rate of biodeterioration (Figure 4).

**Figure 4.** Extended black-greyish patina due to black fungi colonization on marble statue located in the inner yard of the Museum of Messina, Italy, under a tree of *Pittosporum tobira.* Fungal strains isolated from the statue were attributed to meristematic black fungi and genera of *Cladosporium*, *Alternaria* and *Phoma* [81].

The mechanism of biodeterioration of stone monuments caused by black fungi is not fully understood [16,84].

The pattern of colonization of black fungi, and in particular of MCF, demonstrate once more that these microorganisms are well adapted to the stone habitat. In fact, they not only grow on the surface of stone, causing an aesthetic alteration due to the presence of melanin in the mycelium and conidia, described as discoloration, black staining, black spots, and black/greyish patinas [9,20,85]; but they can also act as true endolithic microorganisms by penetrating into the rocks/stones via intercrystalline spaces or through an active mechanism in which both mechanical and chemical aspects are hypothesized.

Microscopic observations show that where they settle is shaped accordingly to the morphology of these fungi (Figure 5). This fact can be explained by a local release of organic acids, followed by a precipitation of mineral phases; this buffer effect may be the reason why these fungi, in contrast to other ubiquitous hyphomycetes, such as *Aspergillus niger* and *Penicillium* spp., as reported by Salvadori and Municchia [16], do not show a marked organic acid production in laboratory conditions. However, Favero Longo et al. [86] demonstrated that some species of MCF (e.g., *Knufia petricola*) penetrate actively into freshly exposed stone probes through the production of iron-chelating molecules (siderophores such as compounds).

Another mechanism is due to the ability of these fungi to penetrate the stone using already existing fractures and cracks. The mechanical forces due to the expansion of hyphae may increase the fractures and cause the loss of materials [18,26,39]. To explain the mechanical penetration of the hyphae into the stone, a past hypothesis gave a crucial role to melanin present in the cell wall of hyphae and in the meristematic cells, similar to the process of penetration of phytopathogenic fungi in the host cells [16,23,25,39].

**Figure 5.** Microscopic observation of the endolithic behaviour of meristematic black fungi. (**a**) Settled on a thin section of Carrara marble sarcophagus flake showing the meristematic chains deepening inside the marble, magnification 400X; (**b**) SEM image showing MCF into the marble with dissolution pattern following the same shape of meristematic cells.

In fact, the melanin present in the appressoria of the phytopathogenic fungi confers turgor and rigidity to the cells, favouring their penetration of the host cells [87].

However, on the basis of the results obtained in a recent publication by Tonon et al. [88], this hypothesis should be rejected. In fact, non-melanized mutants of the black fungus *Knufia petricola* do not lose their ability to penetrate carbonate pellets regardless of porosity; on the contrary, thin, non-melanized, exploring hyphae showed an even higher penetration pattern into the stone, probably due to their nutrient-seeking role and thinness.

#### **4. Multistep Analyses to Study Black Fungi from Stone Monuments**

Contrary to studies carried out on natural rocks, in the frame of cultural heritage it is mandatory to carry out all the necessary multidisciplinary studies (chemical, geological, physical, biological analysis, etc.), by using very low destructive or non-destructive sampling methods [18,46,67,89]. This fact may limit the extent of the studies, but a careful planning of a sampling campaign leads to the right protocol of intervention and of assessing the risk of further biodeterioration processes.

A general useful multistep approach should include microscopy, cultural analyses, molecular analyses, and the laboratory evaluation of selected methods of control (chemical or physical) on isolated strains. The assessment of environmental conditions (temperature, humidity, shining, presence of surrounding vegetation, or other organic sources, atmospheric pollutants, etc.) should be also evaluated. In fact, these data not only allow us a better understanding of the physiology and ecology of fungi, but can help to control their growth indirectly, especially in indoor and/or in confined environments. At the end, a monitoring campaign over time should establish the level of the risk of the item and the frequency of intervention.

Further, a common language for the description of the alterations is also indispensable for sharing the results with the scientific community; to this purpose, there is a glossary [90,91] to obtain an objective and standardized description.

#### *4.1. Methods of Isolation and Characterization*

The sampling is critical. As reported in the previous paragraph, due to the value of the artifacts, non-invasive sampling methods have been developed over the years and are now widely used. Examples are a needle to take samples from black spots or cavities, scalpel or lancet to scrape fungi from the surface, and adhesive tape sample, useful both

for microscopy and cultural and molecular analysis as it provides a mirror image of the stone colonization [22,92,93].

Microscopic examination of the samples is the most common practice to obtain evidence of black fungi directly from stone samples. In fact, due to their size, morphology, and pigmentation, black fungi can be directly visualized under optical microscope without a specific preparation. Even with a good light microscope (LM) or, better, with a scanning electron microscope (SEM), detailed information is obtained. Microscopy is useful for the direct visualization of the fungi in the stone sample, for determining what types of relationship they establish with stone material and with other types of microorganisms, and also for addressing the next step of analyses. Unfortunately, fluorescence microscopy (FM) cannot help in detecting and studying black fungi due to the presence of melanin that masks the fluorochrome fluorescence.

Still, cultural analyses remain the best way to study this group of fungi.

Black fungi, and in particular MCF, possess a poor ability to compete with fast growing fungi, being characterized by a slow growth rate, and very often require more than 1 month of incubation before visible colonies are seen. These reasons explain why they are difficult to isolate and maintain in culture [94]. Nevertheless, selective cultural media that inhibit the growth of bacteria and of fast-growing fungi are successfully employed both for qualitative and quantitative cultural analyses [22,95].

These culture techniques have the advantage of allowing the whole characterization of the isolates by microscopical, biochemical, physiological, and molecular analysis; these latter are indispensable for the identification of MCF that do not have recognizable morphological traits. Multilocus sequencing typing (mlst) is routinely carried out to resolve their taxonomic and phylogenetic position [55,56] by Blast search homology (Basic Local Alignment Search Tools) available online. However, many nucleotide sequences in the Genkank nucleotide database are not updated in the "definition". Therefore, although this approach is within the reach of all laboratories, it requires a deep knowledge of the literature and a curated nucleotide database.

A deeper genetic characterization of the isolates can be obtained by whole genome analyses but, to date, only four genomes belonging to three species of black fungi (*Coniosporium apollinis* CBS 100218, *Knufia petricola* MA5789, *K. petricola* MA5790, and *Aeminium ludgeri* DSM 106916) isolated from stone monuments are available in the NCBI database (https://www.ncbi.nlm.nih.gov/genome/ accessed on 15 February 2022).

Cultural techniques also allow us to investigate the biodeteriorative abilities of the isolated species by setting up laboratory experiments that simulate their settlement, colonization, and biodeterioration pattern [96,97].

#### *4.2. Culture Independent Analyses*

It is well known that only a very small percentage (<0.5%) of environmental microorganisms can be cultured. Therefore, culture independent molecular approaches have been developed to overcome this limitation; the metagenomic approaches allow the study of the microbiota (microbial community) and its microbiome (gene pool) by total acid nucleic extraction from the samples, both DNA and/or RNA, depending on the purpose.

Recently Sterflinger and Pinar [98] reviewed the main molecular-based techniques that are currently available and, although some of these have not yet been used in the field of cultural heritage, they could be adapted for future studies.

In the literature, the majority of papers are focused on Bacteria and Archaea, while there are very few papers about microfungi from stone monuments [99,100].

Large-scale genomic analyses, such as high-throughput-sequencing analyses, are becoming increasingly popular to study the microbiota (both for biodiversity and functional genes analyses) in different areas of research, and cultural heritage is not an exception [89,99]. These techniques were developed in the late 1990s and early 2000s, and today they are more accessible to many laboratories due to the lower costs and the facilities offered by

many companies that can carry out all steps of analysis, from nucleic acid extraction to bioinformatic data analysis.

The culture-independent approach certainly contributes to the deep knowledge on the microorganisms associated with the biodeterioration processes. However, there is a very high risk to obtain a plethora of data that are not easily interpretable, because they cannot be directly connected with the biodeterioration phenomenon observed. In fact, it is obvious, but not trivial, that the discovery of a microbial agent on a monument could be not related to any biodeteriogenic activity. Therefore, the current need is to associate these techniques to the culture-based ones for a more complete characterization of the state of deterioration of the artifact and for implementation of the more suitable prevention strategies.

#### **5. How to Control Black Fungi**

Despite the wide literature regarding the control of biodeteriogens on inorganic surfaces as reported in recent books and reviews [6,9,101–103], very little is said regarding the effectiveness of treatments against black fungi.

Black fungi, especially meristematic ones, are very difficult to eradicate and tend to be one of the first colonizers after cleaning procedures [6,12,83]. In the Lascaux cave, a black yeast *Ochroconis lascauxensis* caused an important and extensive black discoloration on the cave's walls whose origin and evolution were probably linked to the intensive biocide treatments [104].

In order to achieve protection of an artifact, both indirect and direct methods should be implemented. The first ones aim to control, or more realistically to mitigate, the fungal growth by modification of the chemical-physical parameters such as humidity, source of nutrients, and temperature, that are crucial key factors for fungal growth. It is obvious that this is rarely fully achievable, and only in particular circumstances, such as in indoor environments (churches, museums, etc.) or for movable artifacts and objects that can be moved if necessary, while in outdoor conditions this is quite impossible.

Direct treatments aiming to kill/reduce black fungi on the stone should be different on the basis of their colonization pattern (diffuse patina, spot-like colonization, or intercrystalline growth) and on the characteristics of the environment; for example, in an indoor environment, the air is often heavily contaminated by fungal spores and thus they need to be eliminated at the same time as those settled on the surfaces; otherwise, their presence in the air is a continued source of reinfection.

Among the potential methods commonly used to control biodeterioration, physical methods such as mechanical removal and UV and heat shock treatments [101,105], are not very effective against black fungi [102,106].

Regarding chemical methods, in laboratory conditions, classical biocides (e.g., Preventol RI 50, Biotin R, RocimaTM 103) are still the most effective [102,107] and in the field they produce efficient results during cleaning procedures. Plant based extracts show a scarce effectiveness against fungi, and this difficult group of microorganisms is not even taken into account to assess their activity [108]. Nanoparticles are commonly used as biocides due to their activity against algae, cyanobacteria, and most bacteria, but they are not really satisfactory against black fungi.

Protective coatings with antifouling properties may have various effects. In fact, TiO2 based coatings, pure or doped with Ag, show a good effect but are limited to a short/medium term after application [108]. However, in both laboratory and field conditions, after treatments with titania-based coatings, black fungi are the first to recolonize the stone surface in dry environments, while algae first appears in damping walls [63]. Very recently, in laboratory conditions, cholinium@Il based coatings have shown that the use of Il's with a 12 C chains and DBS as anion in combination with nanosilica coatings (e.g., Nano Estel) could be effective against the colonization of black fungi for a period of time over 30 months [109].

One possible explanation of this scarce effectiveness of most treatments against black fungi is that they possess a genetic resistance to environmental stresses, as reported in the previous paragraphs. Therefore, the different mechanisms concurring to the stress protection response may interfere to the biocidal treatments.

Understanding the cause of their resilience could improve the strategies for their control.

#### **6. Concluding Remarks**

The study of biodeterioration of stone monuments is quite complex and cannot be improvised. For a correct understanding of biodeterioration phenomena and the implementation of measures aimed at the elimination and/or mitigation of biodeteriogenic microorganisms, it is important to consider the monument and its surrounding as a whole.

When working for the protection of cultural heritage artifacts, scientists should not follow the same protocols for all the situations. In general, it is necessary to:

The analysis must be planned according to their questions.

It is also important to relate the presence of fungal species with the observed biodeterioration phenomenon; then, for treatments, it is possible to use well known protocols or propose new products/treatments that, however, need to be tested in laboratory with fungal isolates and in situ (on probes, not on the item!!!) before applying it to the CH item. Finally, consider evaluating the use of coatings that match Green conservation criteria and are effective to prevent or slow down new colonization.

As there is not only one method that is valid in all circumstances, we have to work out, case by case, the best solution and monitor the result over the time to avoid unexpected and/or undesirable effects as much as possible.

Black fungi, especially meristematic ones, are very dangerous for stone artifacts for several reasons:

For all these reasons, new methods of study and of control are needed that also aim to search for more eco-friendly molecules and/or approaches.

Despite the increase in interest in black fungi as a cause of biodeterioration of stone monuments and artifacts, many aspects need a more in-depth analysis. For example, not much is known about the molecular mechanisms involved in stress tolerance, in colonization, and biodeterioration of stone. Very important results were achieved by laboratory experiments that, however, concern only some species, and are still too few to generalize the results obtained. Only four genomes of three species were sequenced, and they are not sufficient for a comparative analyses aiming to a better understanding of the above mentioned processes and mechanisms.

Furthermore, the creation of a curated database including the nucleotide sequences used for identification of black fungi from monuments could be very helpful, as the public databases are not curated and are outdated; they could even be misleading for people who are not interested in deepening the knowledge around the taxonomy and phylogeny of this group of fungi.

Finally, it is also important to combine the classical methods with the new ones to improve current knowledge useful for implementation of future conservation strategies.

**Author Contributions:** Conceptualization, F.D.L. and C.U.; methodology, F.D.L., A.M. and C.U.; resources, F.D.L. and C.U.; data curation F.D.L.; writing—original draft preparation, F.D.L., A.M. and C.U. writing—review and editing, F.D.L., A.M. and C.U. All authors have read and agreed to the published version of the manuscript.

**Funding:** No funding was used for this manuscript.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Authors would like to thank Sherron Collins for her revision of the English text.

**Conflicts of Interest:** The authors declare no conflict of interest.

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