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In this study, we explored the biodiversity and abundance of culturable fungi in four samples associated with different biodeterioration outlines collected from the Lemos Pantheon, a limestone-built artwork in Portugal. We compared the results from prolonged standard freezing with those previously obtained from fresh samples to analyze differences in the obtained community and assess the effectiveness of the standard freezing incubation protocol in uncovering a different segment of culturable fungal diversity. Our results showed a slight decrease in culturable diversity, but over 70% of the obtained isolates were not present in the previously studied fresh samples. We also identified a high number of potential new species with this procedure. Moreover, the use of a wide variety of selective culture media positively influenced the diversity of the cultivable fungi obtained in this study. These findings highlight the importance of developing new protocols under varying conditions to accurately characterize the culturable fraction in a given sample. The identification and study of these communities and their possible contribution to the biodeterioration process is crucial knowledge for formulating effective conservation and restoration plans to prevent further damage to valuable cultural heritage assets. [ABSTRACT FROM AUTHOR]

Featured Application: This work expands the current knowledge of fungal agents and their role in the biodeterioration of limestone rocks, while providing tools for the development of better and more adequate strategies for the conservation, restoration, and rehabilitation of the heritage. Although the information presented here is related to a single monument in Portugal, the data can be extrapolated to other monuments of similar lithology. Fungal organisms are considered one of the most relevant stone colonizers, and biodeteriogens. They are ubiquitous heterotrophs, metabolically versatile, ranging from generalist to extremophiles. Limestone, a sedimentary rock characterized by high levels of calcium carbonate, has low compressive strength and hardness and high porosity. These features make it highly susceptible to fungal colonization and an exceptional target for biodeterioration. Understanding the mycobiome composition associated with different biodeterioration scenarios is key for the development of effective guidelines and strategies for preventive conservation and viable maintenance of our cultural heritage. In this work, a thorough analysis of the fungal community composition on the Lemos Pantheon, a limestone-built artwork located in Portugal, was performed using high-throughput sequencing complemented with culture-based methods. The combined results allowed a detailed characterization of the fungal communities of each analyzed spot, revealing highly diverse and dissimilar communities according to the type of biodeterioration observed. In addition, we verified that both cultivation and metagenomics methodologies should be employed synergistically to tackle inherent limitations. [ABSTRACT FROM AUTHOR]

Fungal organisms are considered one of the most relevant stone colonizers, and biodeteriogens. They are ubiquitous heterotrophs, metabolically versatile, ranging from generalist to extremophiles. Limestone, a sedimentary rock characterized by high levels of calcium carbonate, has low compressive strength and hardness and high porosity. These features make it highly susceptible to fungal colonization and an exceptional target for biodeterioration. Understanding the mycobiome composition associated with different biodeterioration scenarios is key for the development of effective guidelines and strategies for preventive conservation and viable maintenance of our cultural heritage. In this work, a thorough analysis of the fungal community composition on the Lemos Pantheon, a limestone-built artwork located in Portugal, was performed using high-throughput sequencing complemented with culture-based methods. The combined results allowed a detailed characterization of the fungal communities of each analyzed spot, revealing highly diverse and dissimilar communities according to the type of biodeterioration observed. In addition, we verified that both cultivation and metagenomics methodologies should be employed synergistically to tackle inherent limitations.