## 4 Discussion  
The protection of cultural heritage often involves the study of the bio-receptivity of building materials and the biodegradation potential of microorganisms involved in deterioration, which can be primarily assessed through laboratory studies. In this work, a non-destructive method was used to sample microorganisms at UNESCO cultural heritage sites in Campania, Italy. In particular, we sampled using adhesive tape sampling coupled with microscopical analysis to identify the constituent microorganisms in biofilms of these monuments, which allows us to examine the existing relationships between the surface and the colonizing microorganisms [9]. The observations made using confocal light microscopy demonstrated fungal colonization in all the adhesive tape samples analyzed as a network of filamentous structures. Furthermore, we observed the presence of cyanobacteria and algae closely connected with filamentous structures, demonstrating that fungi actively colonize the rock as essential compounds of the biofilm sampled and not as contaminants. The isolation of fungi through culture-dependent methods, followed by maintenance of strains in culture, was aimed at obtaining quality-controlled isolates for further studies on biodeterioration processes and to develop innovative strategies for their control. For each strain, we collected data related to the substrate and environmental conditions at the sampling location and this information can be used to develop future laboratory experiments simulating specific environmental conditions under which these fungi can grow. Indeed, future perspectives are directed towards using our fungi as models to perform in vitro experiments for understanding the patterns of microbial colonization of  
Figure 4: The relative prevalence of fungal genera found in: Sibyl Caves in Cuma (**A**), Suburban Baths in Ercolano (**B**), the Roman Amphitheater in Nola (**C**), the House of Poppea in Oplonti (**D**), and the House of Fauno (**E**) and the House of Castricio (**F**) in Pompei. stone materials [21]. The fungal isolates in this study are widespread, frequently associated with soil particles and plant material, which is probably due to their broad tolerance to different environmental conditions and allows them to colonize a large array of terrestrial habitats [27]. Molecular identification of sampled strains was performed using the internal transcribed spacer (ITS) rDNA area, which is the most widely used marker for fungi [19]. Unfortunately, for many Ascomycata genera, such as _Penicillium_ and _Aspergillus_, the ITS is not variable enough to allow for species-level identification [28]. Because of the limitations associated with the chosen molecular marker, herein we considered the species-complex level for an overview of fungal diversity on stone monuments. We recognize the necessity of further identification using taxon-specific markers (e.g., SSRs) for identifying isolates to species level as the collection grows. The genera isolated in our sampling include _Alternaria_, _Aspergillus_, _Cladosporium_, _Clonostachys_, _Curvalaria_, _Fusarium_, _Lecanicillium_, _Neofusicoccum_, _Penicillium_, _Purpurecillium_, _Talaromyces_, and _Trichoderma_, which are known to be ubiquitous filamentous fungi of soil and are often airborne. Some of them were already described as colonizer, or occasionally pioneer, taxa of deteriorated monuments [29]. For most of these species, there is no representative strain of the wide range of morphology and physiology expressed within that species and therefore it is necessary to maintain a number of representative strains. Some collections, such as the CABI Bioscience Genetic Resource Collection, retain on average five strains for each species, but in some cases this number is not sufficient. This is the case for host specificity in the plant pathogen, such as _Fusarium oxysporum_, which has a large number of genetic variants [30].