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Microorganisms naturally colonize rock-based materials in outdoor environments, thereby contributing to their degradation. Fungi, especially in tropical environments with abundant water and favorable temperatures, play a significant role in biodegradation. However, many aspects of the microorganism-stone interaction, including fungal colonization dynamics and the impact of treatment applications, remain unclear. This study conducted a four-year in-situ evaluation of fungal community dynamics on limestone surfaces in the Temple of the Warriors at the Chichén Itzá archaeological site in Mexico, focusing on cleaning and treatment using nanoparticles (NPs). These NPs included zinc oxide (ZnO) and CaZn2(OH)6·2H2O (CZ)-based NPs synthesized via sol-gel (CZ-SG) and mechanochemical methods (CZ-MC), as well as CZ/Ca(OH)2-based products (CZ:Ca-SG). The microbial colonization cover was assessed using colorimetric measurements, and the surface was sampled for fungal community isolation and identification. The results demonstrated significant impacts of cleaning and nanomaterial applications on cultivable fungal communities (melanized filamentous, hyaline, and microcolonial fungi), altering composition, dynamics, and stone surface coloration. In particular, ZnO NPs caused 50 % decline in fungal species and individuals, whereas CZ:Ca-SG NPs displaced most species, indicating effective inhibition of the cultivable fungal community. Microcolonial fungi (MCF), known for their tolerance to withstand harsh environmental conditions, were the only fungal group found in the CZ:Ca-SG treatment. In contrast, CZ-SG and CZ-MC increased the abundance of melanized species, resulting in darkening and reduced color intensity. This study highlights the importance of microcolonial fungi that are tolerant to cleaning and coating procedures in the preservation of stone cultural heritage. These findings enhance our understanding of fungal colonization dynamics following treatment and provide valuable, European Union, CONACYT, Depto. de Mineralogía y Petrología, Fac. de Ciencias Geológicas, TRUE, pub

Background and Aims: The colonization of lithic substrates by fungal communities is determined by the properties of the substrate (bioreceptivity) and climatic and microclimatic conditions. However, the effect of the exposure time of the limestone surface to the environment on fungal communities has not been extensively investigated. In this study, we analyze the composition and structure of fungal communities occurring in biofilms on limestone walls of modern edifications constructed at different times in a subtropical environment in Campeche, Mexico. Methods: A chronosequence of walls built one, five and 10 years ago was considered. On each wall, three surface areas of 3 × 3 cm of the corresponding biofilm were scraped for subsequent analysis. Fungi were isolated by washing and particle filtration technique and were then inoculated in two contrasting culture media (oligotrophic and copiotrophic). The fungi were identified according to macro and microscopic characteristics. Key results: We found 73 genera and 202 species from 844 isolates. Our results showed that fungal communities differed in each biofilm. In the middle-aged biofilm a high number of isolates was found, but both species richness and diversity were low. In contrast, in the old biofilm species richness and diversity were high; Hyphomycete 1, Myrothecium roridum and Pestalotiopsis maculans were abundant. The dominant species in the middle-aged biofilm were Curvularia lunata, Curvularia pallescens, Fusarium oxysporum and Fusarium redolens, and in the young biofilm were Cladosporium cladosporioides, Curvularia clavata, Paraconiothyrium sp. and Phoma eupyrena. Conclusions: Our results suggest that the composition of the fungal community in each biofilm varies according to time of exposure to the environment. Furthermore, the fungal community was composed of a pool of uncommon species that might be autochthonous to limestone., Antecedentes y Objetivos: La colonización de los sustratos líticos por comunidades fúngicas está determinada por las propiedades del sustrato (bioreceptividad) y las condiciones climáticas y microclimáticas. Sin embargo, los efectos del tiempo de exposición de la superficie de la roca calcárea al ambiente sobre la composición de las comunidades fúngicas no se ha investigado. En este estudio, analizamos la composición y estructura de las comunidades fúngicas inmersas en biopelículas asociadas a roca calcárea, en paredes de edificaciones modernas construidas a diferentes tiempos en un ambiente subtropical en Campeche, México. Métodos: Se consideró una cronosecuencia de paredes construidas a uno, cinco y 10 años. Sobre cada pared, se rasparon tres superficies de 3 × 3 cm para cada biopelícula. Los hongos se aislaron por la técnica de lavado y filtración de partículas, posteriormente se inocularon en dos medios de cultivo contrastantes (un medio oligotrófico y uno copiotrófico). Los hongos se identificaron de acuerdo a sus características macro y microscópicas. Resultados clave: Encontramos 73 géneros y 202 especies de 844 aislados. Los resultados mostraron que las comunidades fúngicas son diferentes en las tres biopelículas. En la biopelícula de desarrollo intermedio encontramos un alto número de aislados, pero tanto la riqueza como la diversidad fueron bajas. En contraste, en la biopelícula avanzada, los valores de riqueza de especies y diversidad fueron altos, y las especies abundantes fueron Hyphomycete 1, Myrothecium roridum y Pestalotiopsis maculans. Las especies dominantes en la biopelícula intermedia fueron Curvularia lunata, Curvularia pallescens, Fusarium oxysporum y Fusarium redolens, y en la biopelícula joven fueron Cladosporium cladosporioides, Curvularia clavata, Paraconiothyrium sp. y Phoma eupyrena. Conclusiones: Nuestros resultados sugieren que la composición de la comunidad fúngica en cada biopelícula cambia de acuerdo al tiempo de exposición de la roc