1. Introduction​

Wall paintings are among the most representative elements of figurative artworks and have been developed by human creativity since prehistoric times ^[1]. Their technique of execution requires a layered structure consisting of a support, a ground, and a paint layer, which changed over time across different cultures (secco, such as tempera, or frescoes) ^[2]. In secco technique, which is the earliest, the preparation layers are applied, but the colors remain on the surface, whereas in the frescoes the colors are applied before the mortar dries, allowing their in-depth penetration ^[2]. The employed colours usually have a mineral origin, but some pigments can also be derived from plants. Organic compounds can later be added during restoration or obecause of other human activities (e.g., firing candles in the churches) ^[3].

The observed deterioration phenomena of mural paintings depend largely on the materials used and the environmental conditions ^[4]. Humidity, lighting, temperature, ventilation, and nutrients select the occurring biological agents [1,5]^[1][5]. Organisms belonging to all domains such as bacteria, algae, fungi, arthropods and in a lesser extent lichens, mosses, ferns, and higher plants, have been found on the surfaces of mural paintings [4,7]^[4][6]. Indeed, given the indoor conditions of most mural paintings, photoautotrophs are limited, while fungi and bacteria are more frequent [8] ^[7]. Bacteria with reduced nutritional needs have been often suggested to be the first colonizers. Fungi, due to their large assortment of enzymes and the remarkable ability to thrive in a wide range of environmental conditions, have been recognized as the most common cause of biodeterioration of painted surfaces and other artworks [1,12, 13,14]^{[1][8][9][10]}.

The damage is generally due to the mycelial growth on the substrate, hyphal penetration, and fruiting bodies production onto and into the substrate, all of which increase the volume and number of cracks in the pigment layer and lead to surface fragments detachments [1,15]^[1][11]. Fungal colonization generally starts on the surface and then moves in-depth, up to decreasing painted layer cohesion and cause exfoliations and loss of the paint [9,16]^[12][13]. A study carried out by Dornieden and colleagues demonstrated that some fungi, such as the so-called microcolonial black fungi, are among the most dangerous for cultural heritages and can influence the resistance to shear and torsion stress of mortar and marble, contributing to the separation of different layers of material in mural paintings [17]^[14]. Aesthetic damages are also frequent, due to pigment discolorations, mycelial pigmentation, and/or the release of organic pigments of different colors, depending on the species involved. Extracellular enzymes and/or organic acids are generally released into the substrate from fungal hyphae causing chemical alterations of the mineral constituents of the surfaces as well as the original pigments [9,16]^[12][13]. The secretion of organic acids (e.g., oxalic, citric, succinic, formic, malic, acetic, fumaric, glyoxylic, gluconic, and tartaric acids) can cause dissolution of cations and chelation of metal ions from mortar and mineral pigments, leading to the formation of stable metal complexes whose crystallization causes an increase of internal pressure resulting in cracking, peeling, and the eventual loss of mural fragments [20]^[15].

Awareness of microorganisms' considerable role in preserving art objects and historical buildings dates back to the 1950s [21]^[16]. Ionita and colleagues provided one of the first detailed descriptions of the mycoflora involved in the deterioration of mural paintings of monasteries in Moldavia, noting that it was favoured by the various nutritional sources present in the materials used for the realization of the frescoes and by local environmental parameters [21]^[16]. This was perhaps one of the first statements of the importance of interdisciplinary studies to prevent and control deterioration processes and define restoration and preservation strategies.

Despite the fact that the fungal role in the deterioration of frescoes has been documented by several papers, a global inventory of fungal diversity and their optimal settlement conditions is not yet available. These paintings are mainly present in confined and semi-confined environments, both hypogean and non-hypogean. A fungal alteration pattern dependent on the environmental conditions of these different sites was expected. Those present in hypogean environments are often subjected to a constant extreme humidity, promoting fungal spores germination and mycelial growth. The amount and type of available nutrients also affect the fungal growth rate and the type of fungal taxa. Nutrients may arrive from the external environment as airborne particles, and the more confined are the environments, the lower are the air spores dispersion phenomena. 

2. Fungal Diversity on Wall Paintings

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]^[1][9][10]. 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. Fungi recorded belonged to species found in natural environments like soils, plants, and air. A detailed survey of their diversity and distribution should become a prerequisite before any restoration measures in order to prevent further damages [39] ^[17]. Most records belonged to Ascomycota, with Eurotiales as 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 AlternAlternaria, Fusarium, Cladosporium, Mortierella, Chaetomium, aria, 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]^{[1][18][11][19][17][20][21][22][23][24]}. These taxa are ubiquitous, and their frequent occurrence is due to the production of numerous airborne conidia. A diversity of filamentous fungi, with the most predominant genera Penicillium, Cladosporium, Aspergillus, Penicillium, Cladosporium, Aspergillus, and Trichoderma, were also isolated from mural paintings of the Parish Church of Santo Aleixo (Portugal) [19]^[25].  Species of these genera were recorded on indoor frescoes in Romanian monasteries; their presence was favoured 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]^[26]. Cladosporium species can cope with various harsh environmental conditions thanks to their low nutritional requirements (i.e., in oligotrophic conditions). Otherwise, Chaetomium species are proteolytic and cellulolytic ascomycetes, favoured by nutrient-rich substrates [22,65,117]^[27][28][29]. They were reported as the most frequent microfungi on the frescoes of the St. Damian Monastery in Assisi (Italy) [46]^[20] and on frescoes in a Serbian church [22]^[27]. Furthermore, a community of Aspergillus,Aspergillus, Penicillium, Cladosporium, Penicillium, Cladosporium, and Chaetomium species was recorded from Medieval wall paintings in Styria (Austria), forming spots of different colors [39]^[17]. This group of genera was dominant on two deteriorating frescos in St Clare’s Refectory of the Monastery of St Damian in Assisi [46]^[20].

Hypocreales was the second most abundant order, accounting for 18% of total fungal diversity, within which Acremonium, TrichodermAcremonium, Trichoderma, a, and Fusarium 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]^[30]. Neocosmospora solani Neocosmospora solani, recorded in Thailand, India, Japan, and France; Simplicillium lamellicola Simplicillium lamellicola, recorded in Russia; and Clonostachys rosea [48,58] Clonostachys rosea ^[31][32], recorded in Japan, are examples of mycoparasitic species, while Parengiodontium album  Parengiodontium album is an insect parasite and was recorded in several countries (Germany, Russia, Romania, Austria, Italy, and England) [109]^[33]. The recurrent presence of mites and insects pointed out their possible role in spreading fungi on painted surfaces [46,119]^[20][34].

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]^[22].

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]^[35][36]. A Basidiomycete was also recorded at the entrance of Roman catacombs [121] ^[37], 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 also rarely recorded. 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]^[38][39]. 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]^[40][41][42]. Scolecobasidium lascauxensis and S. anomalus were isolated and described from black stains in Lascaux Cave, France [85,115]^[43][44], while the chaetothyrialean black fungi CladophiCladophialophora, Exophiala, alophora, Exophiala, and Phialophora have been reported from different sites [23,24,42,98] ^{[45][46][47][48]}.

Yeasts have been rarely reported, such as Saccharomycetales (Ascomycota) that usually grow by individual yeast cells or Rhodotorula Rhodotorula spp. (Basidiomycota) often linked to pink/orange stains due to the release of carotenoids [19,93] ^[25][49]. A complete list of species isolated from wall paintings has been reported by Zucconi and colleagues [link with Table 1]