## 4 Discussion  
### 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 _Acemonium_, are among the most common deteriorogens 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 frescoes 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 _Acemonium_, _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 mycopparasites [118]. _Neocosmospora solani_, recorded in Thailand, India, Japan, and France; _Simplicillium lamellicola_, recorded in Russia; and _Clonostachys rose_[48; 58], recorded in Japan, are examples of mycopparasitic 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].