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.

With an impressive ability to survive in harsh environments, black fungi are an ecological group of melanized fungi that are widely recognized as a major contributor to the biodeterioration of stone cultural heritage materials. As part of the ongoing efforts to study the fungal diversity thriving in a deteriorated limestone funerary art piece at the Lemos Pantheon, a national monument located in Águeda, Portugal, two isolates of an unknown microcolonial black fungus were retrieved. These isolates were thoroughly studied through a comprehensive analysis based on a multi-locus phylogeny of a combined dataset of ITS rDNA, LSU, and rpb2, along with morphological, physiological, and ecological characteristics. Based on the data obtained from this integrative analysis, we propose a new genus, Saxispiralis gen. nov., and a new species, Saxispiralis lemnorum sp. nov., in the recently described Aeminiaceae family (order Mycosphaerellales). Prior to this discovery, this family only had one known genus and species, Aeminium ludgeri, also isolated from deteriorated limestone. Additionally, considering the isolation source of the fungus and to better understand its potential contribution to the overall stone monument biodeterioration, its in vitro biodeteriorative potential was also evaluated. This work represents a significant contribution to the understanding of the fungal diversity involved in the biodeterioration of limestone heritage.

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.

Saxispiralis lemnorum MUM 23.14 is an extremotolerant microcolonial black fungus, originally isolated from a biodeteriorated limestone artwork in Portugal. This recently introduced species belongs to the Aeminiaceae family, representing the second member of this monophyletic clade. This fungus exhibits a unique set of characteristics, including xerophily, cold tolerance, high UV radiation tolerance, and an exceptional ability to thrive in NaCl concentrations of up to 30% while also enduring pH levels ranging from 5 to 11. To gain insights into its genomic traits associated with stress resistance mechanisms, specialization, and their potential implications in stone biodeterioration, we conducted a comprehensive genome sequencing and analysis. This draft genome not only marks the first for the Saxispiralis genus but also the second for the Aeminiaceae family. Furthermore, we performed two comparative genomic analyses: one focusing on the closest relative within the Aeminiaceae family, Aeminium ludgeri, and another encompassing the genome of different extremotolerant black fungi. In this study, we successfully achieved high genome completeness for S. lemnorum and confirmed its close phylogenetic relationship to A. ludgeri. Our findings revealed traits contributing to its extremophilic nature and provided insights into potential mechanisms contributing to stone biodeterioration. Many traits are common to both Aeminiaceae species and are shared with other black fungi, while numerous unique traits may be attributed to species-specific characteristics.

Fusarium circinatum, the causative agent of pine pitch canker disease, is a pathogenic fungus that poses a significant threat to pine forests globally. It infects various Pinus species, causing resinous cankers, needle discoloration, and tree death. The disease severely impacts forest ecosystems, necessitating cost-effective and environmentally friendly management strategies. Contaminated pine seeds and seedlings are the main pathways for introducing this fungus to disease-free areas. To mitigate this disease and prevent its spread, it is crucial to implement new processes in forest plant production systems that align with the existing conditions of forest nurseries, ensuring effective and sustainable management. With this in mind, a national collaborative study involving 14 Portuguese partners was initiated to develop new prevention and mitigation strategies. In this work, four different treatments—MennoFlorades, Captan, ethanol, and hot water—were tested for their ability to eliminate F. circinatum from contaminated Pinus seeds in vitro. The most effective treatments were selected for further in vitro assays and real-context nursery germination trials to assess their impacts on seed germination, plant production, and certification. MennoFlorades, Captan, and hot water were tested in the nursery, with hot water showing the most promising results due to its negligible impact on seedlings, eco-friendly nature, ease of implementation, and cost-effectiveness. These findings offer promising prospects for preventing pine pitch canker outbreaks in nurseries and, consequently, in forests.

As is the case for all forms of cultural heritage, museum collections are under a serious threat of biodeterioration, with fungi playing a significant role in this phenomenon, compromising the preservation and dissemination of past knowledge, artifacts and traditions. As part of the ongoing efforts to study the fungal contaminations observed in the artifacts that compose the collection of the Science Museum of the University of Coimbra, non-invasive sampling of four objects present in the archive was conducted and 43 fungal isolates were retrieved. These isolates were subjected to a comprehensive analysis based on molecular and morphological characteristics and were identified as belonging to 15 different species. Of the 15 isolated species, 5 were selected for in vitro antifungal essential oil assays, based on their relative abundance and biodeteriorative potential. For these, essential oils of Cymbopogon citratus, Lavandula angustifolia, Thapsia gummifera, Mentha pulegium, Mentha suaveolens, Thapsia villosa, Thymus mastichina and Agastache foeniculum were obtained via hydrodistillation, in accordance with the European Pharmacopeia. Of the tested oils, most impacted fungal growth, with Cymbopogon citratus and Lavandula angustifolia being able to fully inhibit the growth of most fungi even at the lowest tested concentrations.