Once upon a Time, There Was a Piece of Wood: Present Knowledge and Future Perspectives in Fungal Deterioration of Wooden Cultural Heritage in Terrestrial Ecosystems and Diagnostic Tools.

Wooden Cultural Heritage (WCH) represents a significant portion of the world's historical and artistic heritage, consisting of immovable and movable artefacts. Despite the expertise developed since ancient times to enhance its durability, wooden artefacts are inevitably prone to degradation. Fungi play a pivotal role in the deterioration of WCH in terrestrial ecosystems, accelerating its decay and leading to alterations in color and strength. Reviewing the literature of the last 25 years, we aimed to provide a comprehensive overview of fungal diversity affecting WCH, the biochemical processes involved in wood decay, and the diagnostic tools available for fungal identification and damage evaluation. Climatic conditions influence the occurrence of fungal species in threatened WCH, characterized by a prevalence of wood-rot fungi (e.g., Serpula lacrymans, Coniophora puteana) in architectural heritage in temperate and continental climates and Ascomycota in indoor and harsh environments. More efforts are needed to address the knowledge fragmentation concerning biodiversity, the biology of the fungi involved, and succession in the degradative process, which is frequently centered solely on the main actors. Multidisciplinary collaboration among engineers, restorers, and life sciences scientists is vital for tackling the challenges posed by climate change with increased awareness. Traditional microbiology and culture collections are fundamental in laying solid foundations for a more comprehensive interpretation of big data.

Trends in Antarctic soil fungal research in the context of environmental changes.

Antarctic soils represent one of the most pristine environments on Earth, where highly adapted and often endemic microbial species withstand multiple extremes. Specifically, fungal diversity is extremely low in Antarctic soils and species distribution and diversity are still not fully characterized in the continent. Despite the unique features of this environment and the international interest in its preservation, several factors pose severe threats to the conservation of inhabiting ecosystems. In this light, we aimed to provide an overview of the effects on fungal communities of the main changes endangering the soils of the continent. Among these, the increasing human presence, both for touristic and scientific purposes, has led to increased use of fuels for transport and energy supply, which has been linked to an increase in unintentional environmental contamination. It has been reported that several fungal species have evolved cellular processes in response to these soil contamination episodes, which may be exploited for restoring contaminated areas at low temperatures. Additionally, the effects of climate change are another significant threat to Antarctic ecosystems, with the expected merging of previously isolated ecosystems and their homogenization. A possible reduction of biodiversity due to the disappearance of well-adapted, often endemic species, as well as an increase of biodiversity, due to the spreading of non-native, more competitive species have been suggested. Despite some studies describing the specialization of fungal communities and their correlation with environmental parameters, our comprehension of how soil communities may respond to these changes remains limited. The majority of studies attempting to precisely define the effects of climate change, including in situ and laboratory simulations, have mainly focused on the bacterial components of these soils, and further studies are necessary, including the other biotic components.

The hidden network of biocrust successional stages in the High Arctic: Revealing abiotic and biotic factors shaping microbial and metazoan communities.

Despite the important role that biocrust communities play in maintaining ecosystem structure and functioning in deglaciated barren soil, few studies have been conducted on the dynamics of biotic communities and the impact of physicochemical characteristics in shaping the different successional stages. In this study an integrated approach encompassing physicochemical parameters and molecular taxonomy was used for identifying the indicator taxa and the presence of intra- and inter-kingdom interactions in five different crust/biocrust successional stages: i) physical crust, ii) cyanobacteria-dominated biocrust, iii) cyanobacteria/moss-dominated biocrust, iv) moss-dominated biocrust and v) bryophyte carpet. The phylum Gemmatimonadota was the bacterial indicator taxon in the early stage, promoting both inter- and intra-kingdom interactions, while Cyanobacteria and Nematoda phyla played a pivotal role in formation and dynamics of cyanobacteria-dominated biocrusts. A multitrophic community, characterized by a shift from oligotrophic to copiotrophic bacteria and the presence of saproxylic arthropod and herbivore insects was found in the cyanobacteria/moss-dominated biocrust, while a more complex biota, characterized by an increased fungal abundance (classes Sordariomycetes, Leotiomycetes, and Dothideomycetes, phylum Ascomycota), associated with highly trophic consumer invertebrates (phyla Arthropoda, Rotifera, Tardigrada), was observed in moss-dominated biocrusts. The class Bdelloidea and the family Hypsibiidae (phyla Rotifera and Tardigrada, respectively) were metazoan indicator taxon in bryophyte carpet, suggesting their potential role in shaping structure and function of this late successional stage. Nitrogen and phosphorus were the main physicochemical limiting factors driving the shift among different crust/biocrust successional stages. Identification and characterization of indicator taxa, biological intra- and inter-kingdom interactions and abiotic factors driving the shift among different crust/biocrust successional stages provide a detailed picture on crust/biocrust dynamics, revealing a strong interconnection among micro- and macrobiota systems. These findings enhance our understanding of biocrust ecosystems in High Arctic, providing valuable insights for their conservation and management in response to environmental shifts due to climate change.

Recolonization dynamics of marble monuments after cleaning treatments: A nine-year follow-up study.

The prevention and control of biological patinas on outdoor stone monuments represent a demanding challenge for the conservation of cultural heritage also due to some microorganisms, particularly resistant to treatments, such as black meristematic fungi, an eco-physiological group well known for its tolerance to extreme conditions. Even if several methods and eco-friendly products have been proposed as new alternatives, traditional biocides are still far from being completely replaced. Recolonization is a natural process that occurs sooner or later after cleaning. The time that elapses until its occurrence can vary considerably depending on environmental conditions and the used products; unfortunately, the papers describing the effect of treatments over time are rare. This work aims to shed light on the recolonization process of marble surfaces in the ancient monumental cemetery of Bonaria (Cagliari) after nine years from treatments, evaluating the long-term efficiency of two different cleaning methods, namely dimethyl sulfoxide-based gel (DMSO-based gel) and Biotin T (a didecyldimethylammonium chloride-based product-). In this context, the microflora present before treatments and in the following years was assessed by culture-based methods and identified by molecular techniques, with attention on black meristematic fungi, which were used as reference for the most resistant lithobiontic organisms. Different environmental parameters, such as temperature, exposition, dominant winds, and rainfall, were considered, and infrared thermography, portable light microscopy, and image analysis were used. This research evidenced the influence of water availability and lightning in recolonization processes, the transition from the pioneer fungal community versus more resistant black fungal species after Biotin T treatment, and the long-lasting efficiency of the DMSO-based gel. These findings prove that this low-impact method deserves more attention in the conservation of outdoor marble monuments, emphasizing the importance of long-term studies.

Wide divergence of fungal communities inhabiting rocks and soils in a hyper-arid Antarctic desert.

Highly simplified microbial communities colonise rocks and soils of continental Antarctica ice-free deserts. These two habitats impose different selection pressures on organisms, yet the possible filtering effects on the diversity and composition of microbial communities have not hitherto been fully characterised. We hence compared fungal communities in rocks and soils in three localities of inner Victoria Land. We found low fungal diversity in both substrates, with a mean species richness of 28 across all samples, and significantly lower diversity in rocks than in soils. Rock and soil communities were strongly differentiated, with a multinomial species classification method identifying just three out of 328 taxa as generalists with no affinity for either substrate. Rocks were characterised by a higher abundance of lichen-forming fungi (typically Buellia, Carbonea, Pleopsidium, Lecanora, and Lecidea), possibly owing to the more protected environment and the porosity of rocks permitting photosynthetic activity. In contrast, soils were dominated by obligate yeasts (typically Naganishia and Meyerozyma), the abundances of which were correlated with edaphic factors, and the black yeast Cryomyces. Our study suggests that strong differences in selection pressures may account for the wide divergences of fungal communities in rocks and soils of inner Victoria Land.

Draft genome sequencing of Naganishia species isolated from the polar environments.

The draft genomes of five Naganishia strains were sequenced using MinION and annotated using Funannotate pipeline. Phylogenetic and genomic analyses were performed to provide their genetic relationships, diversity, and potential functional capabilities. This approach will aid in understanding their potential to survive under microgravity and their resilience to extreme environments.

Antarctic Soil Metabolomics: A Pilot Study.

In Antarctica, ice-free areas can be found along the coast, on mountain peaks, and in the McMurdo Dry Valleys, where microorganisms well-adapted to harsh conditions can survive and reproduce. Metabolic analyses can shed light on the survival mechanisms of Antarctic soil communities from both coastal sites, under different plant coverage stages, and inner sites where slow-growing or dormant microorganisms, low water availability, salt accumulation, and a limited number of primary producers make metabolomic profiling difficult. Here, we report, for the first time, an efficient protocol for the extraction and the metabolic profiling of Antarctic soils based on the combination of NMR spectroscopy and mass spectrometry (MS). This approach was set up on samples harvested along different localities of Victoria Land, in continental Antarctica, devoid of or covered by differently developed biological crusts. NMR allowed for the identification of thirty metabolites (mainly sugars, amino acids, and organic acids) and the quantification of just over twenty of them. UPLC-MS analysis identified more than twenty other metabolites, in particular flavonoids, medium- and long-chain fatty acids, benzoic acid derivatives, anthracenes, and quinones. Our results highlighted the complementarity of the two analytical techniques. Moreover, we demonstrated that their combined use represents the "gold standard" for the qualitative and quantitative analysis of little-explored samples, such as those collected from Antarctic soils.

Effects of 5-year experimental warming in the Alpine belt on soil Archaea: Multi-omics approaches and prospects.

We currently lack a predictive understanding of how soil archaeal communities may respond to climate change, particularly in Alpine areas where warming is far exceeding the global average. Here, we characterized the abundance, structure, and function of total (by metagenomics) and active soil archaea (by metatranscriptomics) after 5-year experimental field warming (+1°C) in Italian Alpine grasslands and snowbeds. Our multi-omics approach unveiled an increasing abundance of Archaea during warming in snowbeds, which was negatively correlated with the abundance of fungi (by qPCR) and micronutrients (Ca and Mg), but positively correlated with soil water content. In the snowbeds transcripts, warming resulted in the enrichment of abundances of transcription and nucleotide biosynthesis. Our study provides novel insights into possible changes in soil Archaea composition and function in the climate change scenario.

Knufia obscura sp. nov. and Knufia victoriae sp. nov., two new species from extreme environments.

Six strains of black meristematic fungi were isolated from Antarctic soils, gasoline car tanks and from the marine alga Flabellia petiolata. These fungi were characterized by morphological, physiological and phylogenetic analyses. According to the maximum-likelihood analysis reconstructed with ITS and LSU sequences, these strains belonged to the genus Knufia. Knufia obscura sp. nov. (holotype CBS 148926) and Knufia victoriae sp. nov. (holotype CBS 149015) are proposed as two novel species and descriptions of their morphological, physiological and phylogenetic features are presented. Based on the maximum-likelihood analyses, K. obscura was closely related to Knufia hypolithi (99 % bootstrap support), while K. victoriae clustered in the clade of Knufia cryptophialidica and Knufia perfecta (93 % bootstrap support). Knufia victoriae, recorded in Antarctic soil samples, had a psychrophilic behaviour, with optimal growth between 10 and 15 °C and no growth recorded at 20 °C. Knufia obscura, from a gasoline car tank and algae, displayed optimal growth between 20 and 25 °C and was more tolerant to salinity than K. victoriae.

Biosignature stability in space enables their use for life detection on Mars.

Two rover missions to Mars aim to detect biomolecules as a sign of extinct or extant life with, among other instruments, Raman spectrometers. However, there are many unknowns about the stability of Raman-detectable biomolecules in the martian environment, clouding the interpretation of the results. To quantify Raman-detectable biomolecule stability, we exposed seven biomolecules for 469 days to a simulated martian environment outside the International Space Station. Ultraviolet radiation (UVR) strongly changed the Raman spectra signals, but only minor change was observed when samples were shielded from UVR. These findings provide support for Mars mission operations searching for biosignatures in the subsurface. This experiment demonstrates the detectability of biomolecules by Raman spectroscopy in Mars regolith analogs after space exposure and lays the groundwork for a consolidated space-proven database of spectroscopy biosignatures in targeted environments.

Fungal communities in European alpine soils are not affected by short-term in situ simulated warming than bacterial communities.

The impact of global warming on biological communities colonizing European alpine ecosystems was recently studied. Hexagonal open top chambers (OTCs) were used for simulating a short-term in situ warming (estimated around 1°C) in some alpine soils to predict the impact of ongoing climate change on resident microbial communities. Total microbial DNA was extracted from soils collected either inside or outside the OTCs over 3 years of study. Bacterial and fungal rRNA copies were quantified by qPCR. Metabarcoding sequencing of taxonomy target genes was performed (Illumina MiSeq) and processed by bioinformatic tools. Alpha- and beta-diversity were used to evaluate the structure of bacterial and fungal communities. qPCR suggests that, although fluctuations have been observed between soils collected either inside and outside the OTCs, the simulated warming induced a significant (p < 0.05) shift only for bacterial abundance. Likewise, significant (p < 0.05) changes in bacterial community structure were detected in soils collected inside the OTCs, with a clear increase of oligotrophic taxa. On the contrary, fungal diversity of soils collected either inside and outside the OTCs did not exhibit significant (p < 0.05) differences, suggesting that the temperature increase in OTCs compared to ambient conditions was not sufficient to change fungal communities.

Composition and functioning of the soil microbiome in the highest altitudes of the Italian Alps and potential effects of climate change.

As the European Alps are experiencing a strong climate warming, this study analyzed the soil microbiome at different altitudes and among different vegetation types at the Stelvio Pass (Italian Alps), aiming to (i) characterize the composition and functional potential of the microbiome of soils and their gene expression during the peak vegetative stage; (ii) explore the potential short-term (using open-top chambers) and long-term (space-for-time substitutions) effects of increasing temperature on the alpine soil microbiome. We found that the functional potential of the soil microbiome and its expression differed among vegetation types. Microbial α-diversity increased along the altitudinal gradient. At lower altitude, shrubland had the highest proportion of fungi, which was correlated with higher amounts of CAZymes, specific for degrading fungal biomass and recalcitrant plant biopolymers. Subalpine upward vegetation shift could lead a possible loss of species of alpine soils. Shrub encroachment may accelerate higher recalcitrant C decomposition and reduce total ecosystem C storage, increasing the efflux of CO2 to the atmosphere with a positive feedback to warming. A total of 5 years of warming had no effect on the composition and functioning of microbial communities, indicating that longer-term warming experiments are needed to investigate the effects of temperature increases on the soil microbiome.

Hydrocarbon-Contaminated Sites: Is There Something More Than Exophiala xenobiotica? New Insights into Black Fungal Diversity Using the Long Cold Incubation Method.

Human-made hydrocarbon-rich environments are important reservoirs of microorganisms with specific degrading abilities and pathogenic potential. In particular, black fungi are of great interest, but their presence in the environment is frequently underestimated because they are difficult to isolate. In the frame of a biodiversity study from fuel-contaminated sites involving 30 diesel car tanks and 112 fuel pump dispensers (52 diesel and 60 gasoline, respectively), a total of 181 black fungal strains were isolated. The long cold incubation (LCI) of water-suspended samples, followed by plating on Dichloran Rose Bengal Chloramphenicol Agar (DRBC), gave isolation yields up to six times (6.6) higher than those of direct plating on DRBC, and those of enrichment with a phenolic mix. The sequencing of ITS and LSU-rDNA confirmed the dominance of potentially pathogenic fungi from the family Herpotrichiellaceae and Exophiala xenobiotica. Moreover, other opportunistic species were found, including E. opportunistica, E. oligosperma, E. phaeomuriformis, and Rhinocladiella similis. The recurrent presence of E. crusticola, Knufia epidermidis, Aureobasidium melanogenum, Cladosporium spp., and Scolecobasidium spp. was also recorded. Interestingly, 12% of total isolates, corresponding to 50% of taxa found (16/32), represent new species. All the novel taxa in this study were isolated by LCI. These findings suggest that black fungal diversity in hydrocarbon-rich niches remains largely unexplored and that LCI can be an efficient tool for further investigations.

Dark-pigmented biodeteriogenic fungi in etruscan hypogeal tombs: New data on their culture-dependent diversity, favouring conditions, and resistance to biocidal treatments.

Subterranean Cultural Heritage sites are frequently subject to biological colonization due to the high levels of humidity, even in conditions of low irradiance and oligotrophy. Here microorganisms form complex communities that may be dangerous through mineral precipitation, through the softening of materials or causing frequent surface discolorations. A reduction of contamination's sources along with the control of microclimatic conditions and biocide treatments (overall performed with benzalkonium chloride) are necessary to reduce microbial growths. Dark discolorations have been recorded in the painted Etruscan tombs of Tarquinia, two of which have been analyzed to collect taxonomical, physiological, and ecological information. Eighteen dark-pigmented fungi were isolated among a wider culturable fraction: nine from blackening areas and nine from door sealings, a possible route of contamination. Isolates belonged to three major groups: Chaetothyriales, Capnodiales (Family Cladosporiaceae), and Acremonium-like fungi. Exophiala angulospora and Cyphellophora olivacea, a novelty for hypogea, were identified, while others need further investigations as possible new taxa. The metabolic skills of the detected species showed their potential dangerousness for the materials. Their tolerance to benzalkonium chloride-based products suggested a certain favouring effect through the decreasing competitiveness of less resistant species. The type of covering of the dromos may influence the risk of outer contamination. Fungal occurrence can be favoured by root penetration.

Plant DNA Barcode as a Tool for Root Identification in Hypogea: The Case of the Etruscan Tombs of Tarquinia (Central Italy).

Roots can produce mechanical and chemical alterations to building structures, especially in the case of underground historical artifacts. In archaeological sites, where vegetation plays the dual role of naturalistic relevance and potential threat, trees and bushes are under supervision. No customized measures can be taken against herbaceous plants lacking fast and reliable root identification methods that are useful to assess their dangerousness. In this study, we aimed to test the efficacy of DNA barcoding in identifying plant rootlets threatening the Etruscan tombs of the Necropolis of Tarquinia. As DNA barcode markers, we selected two sections of the genes rbcL and matK, the nuclear ribosomal internal transcribed spacer (nrITS), and the intergenic spacer psbA-trnH. All fourteen root samples were successfully sequenced and identified at species (92.9%) and genus level (7.01%) by GenBank matching and reference dataset implementation. Some eudicotyledons with taproots, such as Echium italicum L., Foeniculum vulgare Mill., and Reseda lutea L. subsp. lutea, showed a certain recurrence. Further investigations are needed to confirm this promising result, increasing the number of roots and enlarging the reference dataset with attention to meso-Mediterranean perennial herbaceous species. The finding of herbaceous plants roots at more than 3 m deep confirms their potential risk and underlines the importance of vegetation planning, monitoring, and management on archaeological sites.

Highly differentiated soil bacterial communities in Victoria Land macro-areas (Antarctica).

Ice-free areas of Victoria Land, in Antarctica, are characterized by different terrestrial ecosystems, that are dominated by microorganisms supporting highly adapted communities. Despite the unique conditions of these ecosystems, reports on their bacterial diversity are still fragmentary. From this perspective, 60 samples from 14 localities were analyzed. These localities were distributed in coastal sites with differently developed biological soil crusts, inner sites in the McMurdo Dry Valleys with soils lacking of plant coverage, and a site called Icarus Camp, with a crust developed on a thin locally weathered substrate of the underlying parent granitic-rock. Bacterial diversity was studied through 16S rRNA metabarcoding sequencing. Communities diversity, composition and the abundance and composition of different taxonomic groups were correlated to soil physicochemical characteristics. Firmicutes, Bacteroidetes, Cyanobacteria and Proteobacteria dominated these communities. Most phyla were mainly driven by soil granulometry, an often disregarded parameter and other abiotic parameters. Bacterial composition differed greatly among the three macrohabitats, each having a distinct bacterial profile. Communities within the two main habitats (coastal and inner ones) were well differentiated from each other as well, therefore depending on site-specific physicochemical characteristics. A core community of the whole samples was observed, mainly represented by Firmicutes and Bacteroidetes.

Growth Forms and Functional Guilds Distribution of Soil Fungi in Coastal Versus Inland Sites of Victoria Land, Antarctica.

In Victoria Land, Antarctica, ice-free areas are restricted to coastal regions and dominate the landscape of the McMurdo Dry Valleys. These two environments are subjected to different pressures that determine the establishment of highly adapted fungal communities. Within the kingdom of fungi, filamentous, yeasts and meristematic/microcolonial growth forms on one side and different lifestyles on the other side may be considered adaptive strategies of particular interest in the frame of Antarctic constraints. In this optic, soil fungal communities from both coastal and Dry Valleys sites, already characterized thorough ITS1 metabarcoding sequencing, have been compared to determine the different distribution of phyla, growth forms, and lifestyles. Though we did not find significant differences in the richness between the two environments, the communities were highly differentiated and Dry Valleys sites had a higher evenness compared to coastal ones. Additionally, the distribution of different growth forms and lifestyles were well differentiated, and their diversity and composition were likely influenced by soil abiotic parameters, among which soil granulometry, pH, P, and C contents were the potential main determinants.

Microbial activity in alpine soils under climate change.

Soil enzymatic activity was assessed in the Stelvio Pass area (Italian Central Alps) aiming to define the possible effects of climate change on microbial functioning. Two sites at two different elevations were chosen, a subalpine (2239 m) and an alpine belt (2604-2624 m), with mean annual air temperature differing by almost 3 °C, coherent with the worst future warming scenario (RCP 8.5) by 2100. The lower altitude site may represent a proxy of the potential future situation at higher altitude after the upward shift of subalpine vegetation due to climate change. Additionally, hexagonal open top chambers (OTCs) were installed at the upper site, to passively increase by about 2 °C the summer inner temperature to simulate short term effects of warming before the vegetation shift takes place. Soil physicochemical properties and the bacterial and fungal abundances of the above samples were also considered. The subalpine soils showed a higher microbial activity, especially for hydrolytic enzymes, higher carbon, ammonium and hydrogen (p < 0.001) contents, and a slightly higher PO4 content (p < 0.05) than alpine soils. Bacterial abundance was higher than fungal abundance, both for alpine and subalpine soils. On the other hand, the short term effect, which increased the mean soil temperature during the peak of the growing season in the OTC, showed to induce scarcely significant differences for edaphic parameters and microbial biomass content among the warmed and control plots. Using the manipulative warming experiments, we demonstrated that warming is able to change the enzyme activity starting from colder and higher altitude sites, known to be more vulnerable to the rising temperatures associated with climate change. Although five-years of experimental warming does not allow us to make bold conclusions, it appeared that warming-induced upwards vegetation shift might induce more substantial changes in enzymatic activities than the short-term effects, in the present vegetation context.