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.

The Metallotolerance and Biosorption of As(V) and Cr(VI) by Black Fungi.

A collection of 34 melanized fungi isolated previously from anthropogenic contaminated sites were assessed for their tolerance to toxic concentrations of As(V) and Cr(VI) anions. Three strains of the species Cyphellophora olivacea, Rhinocladiella similis, and Exophiala mesophila (Chaetothyriales) were identified as hyper-metallotolerant, with estimated IC50 values that ranged from 11.2 to 16.9 g L-1 for As(V) and from 2.0 to 3.4 g L-1 for Cr(VI). E. mesophila and R. similis were selected for subsequent assays on their biosorption capacity and kinetics under different pH values (4.0 and 6.5) and types of biomass (active and dead cells and melanin extracts). The fungal biosorption of As(V) was relatively ineffective, but significant removal of Cr(VI) was observed from liquid cultures. The Langmuir model with second-order kinetics showed maximum sorption capacities of 39.81 mg Cr6+ g-1 for R. similis and 95.26 mg Cr6+ g-1 for E. mesophila on a dry matter basis, respectively, while the kinetic constant for these two fungi was 1.32 × 10-6 and 1.39 × 10-7 g (mg Cr6+ min)-1. Similar experiments with melanin extracts of E. mesophila showed maximum sorption capacities of 544.84 mg Cr6+ g-1 and a kinetic constant of 1.67 × 10-6 g (mg Cr6+ min)-1. These results were compared to bibliographic data, suggesting that metallotolerance in black fungi might be the result of an outer cell-wall barrier to reduce the diffusion of toxic metals into the cytoplasm, as well as the inner cell wall biosorption of leaked metals by melanin.

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.

The Roman Houses of the Caelian Hill (Rome, Italy): Multitemporal Evaluation of Biodeterioration Patterns.

Like other hypogeal environments, the Roman Houses of the Caelian Hill are prone to unwanted biological growth. Wide conservative interventions have been carried out at the beginning of this millenium to reduce biodeterioration and physical-chemical damages. Retracing the last monitoring work, we assessed the site's current state of conservation and biodeterioration intending to check the previous treatments' effectiveness and deepen the common knowledge of the subterranean biota and their possible biodeteriogenic effects. Starting from the past test areas and the previous identifications of the occurring biodeteriogens, we further isolated and identified the main eubacterial, fungal, and phototrophic settlers, focusing on some detrimental traits for wall paintings (i.e., acid production and carbonate precipitation). The achieved results proved the success of the performed interventions in reducing the wall's water content. Otherwise, the new conditions raise, in the long term, new concerns about lampenflora, carbonate precipitations, and salt efflorescence. Here, the Caelian Houses' new status is documented. The possible favouring conditions for the different groups of biodeteriogens, along with the taxonomical novelties, additional risks tied to the anthropization of the resident culturable microbial community, and the possible relation between the black fungus Cyphellophora olivacea and roots, are reported and discussed.

Science for art: multi-years' evaluations of biocidal efficacy in support of artwork conservation.

In recent decades, the relationship between Science and Art has been gradually strengthened through the use of diagnostic, conservation, and valorization technologies. New technologies can also be used to support the creation and durability of bio-artworks. Within such a context, starting from the Spring of 2014, we performed in situ experimentations to eventually increase the durability of the graphical artwork of William Kentridge on the Lungotevere embankments, whose creation was scheduled in the following years. We applied various combinations and concentrations of three different biocides (Algophase, Biotin R, and Preventol R80) and two water repellents (Hydrophase surfaces and Silo 111) on 34 test areas. However, the artist preferred to leave his artwork to a natural fading. Right before the realization of the graphical artwork "Triumph and Laments of Rome" in 2016, just the black biological colonizations mainly composed of cyanobacteria were removed through pressurized water. We monitored the artist's work through analyses of images and colorimetric variations and such drawings showed a duration of 4 years in the natural conditions of recolonization. Here we show how the recolonization of treated and control areas, analyzed with the same methods, showed an increased duration, 3 years longer than under natural conditions in the case of Preventol R80® and Biotin R® plus Silo 111®. The tested solutions showed differential effectiveness and multiple possibilities of use to support the maintenance of the artwork if the artist wanted to preserve his artwork for a longer period.

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.

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.

Rock black fungi: excellence in the extremes, from the Antarctic to space.

This work focuses on rock-inhabiting fungi (RIF) of Antarctic rocky deserts, considered the closest to a possible Martian habitat, as the best example of adaptation to the extremes. The study of RIF ecophysiology, resistance and adaptation provides tools that shed light on the evolution of extremophily. These studies also help define the actual limits for life and provide insight for investigating its existence beyond our planet. The scientific results obtained from over 20 years of research on the biodiversity, phylogeny and evolution toward extremotolerance reviewed here demonstrate how these fascinating organisms can withstand conditions well beyond those in their natural environment. A final focus is given on results and perspectives arising from a recent proteomic approach, and from astrobiological experiments and their significance for future space exploration. These studies demonstrate that Antarctic RIF offer an excellent opportunity to investigate many basic, but also applicative areas of research on extremophily.

Bradymyces gen. nov. (Chaetothyriales, Trichomeriaceae), a new ascomycete genus accommodating poorly differentiated melanized fungi.

Three slow growing, melanized and morphologically poorly differentiated fungal strains were isolated from a hyperaemic focus near the enlarged spleen of a farmed rainbow trout (Oncorhynchus mykiss) and from a rock collected at 3,200 m a. s. l. (Alps, Italy). Two phylogenetic analyses of the combined nuc18S and nuc28S rDNA and ITS rDNA and ß-tubulin sequences showed that these isolates belong to the Trichomeriaceae, a family of the ascomycete order Chaetothyriales containing black yeasts that cause infections in humans and animals. The strains form a well-supported monophyletic clade. The new genus Bradymyces, with two new species, Bradymyces oncorhynchi and Bradymyces alpinus, is proposed based on phylogenetic, ecophysiological and morphological data. It is characterized by the presence of moniliform hyphae, blastic proliferation, endoconidia, multicellular and muriform bodies, and bodies with dark fragmented incrustations on the surface. Bradymyces most closely resembles members of Knufia. The ex-type isolate of B. oncorhynchi CCF 4369(T) ( = CBS 133066(T) = CCFEE 6134(T)) represents the first case of a Trichomeriaceae member isolated from cold-blooded water vertebrates. B. alpinus [ex-type strain CCFEE 5493(T) ( = CBS 138368(T) = CCF 4803(T))] is represented by two isolates from a single locality in the Alps and in contrast to B. oncorhynchi shows overall slower growth parameters and does not grow at 25 °C.

Description of Taphrina antarctica f.a. sp. nov., a new anamorphic ascomycetous yeast species associated with Antarctic endolithic microbial communities and transfer of four Lalaria species in the genus Taphrina.

In the framework of a large-scale rock sampling in Continental Antarctica, a number of yeasts have been isolated. Two strains that are unable to grow above 20 °C and that have low ITS sequence similarities with available data in the public domain were found. The D1/D2 LSU molecular phylogeny placed them in an isolated position in the genus Taphrina, supporting their affiliation to a not yet described species. Because the new species is able to grow in its anamorphic state only, the species Taphrina antarctica f.a. (forma asexualis) sp. nov. has been proposed to accommodate both strains (type strain DBVPG 5268(T), DSM 27485(T) and CBS 13532(T)). Lalaria and Taphrina species are dimorphic ascomycetes, where the anamorphic yeast represents the saprotrophic state and the teleomorph is the parasitic counterpart on plants. This is the first record for this genus in Antarctica; since plants are absent on the continent, we hypothesize that the fungus may have focused on the saprotrophic part of its life cycle to overcome the absence of its natural host and adapt environmental constrains. Following the new International Code of Nomenclature for Algae, Fungi and Plants (Melbourne Code 2011) the reorganization of Taphrina-Lalaria species in the teleomorphic genus Taphrina is proposed. We emend the diagnosis of the genus Taphrina to accommodate asexual saprobic states of these fungi. Taphrina antarctica was registered in MycoBank under MB 808028.

The mTOR signaling pathway and neuronal stem/progenitor cell proliferation in the hippocampus are altered during the development of absence epilepsy in a genetic animal model.

Hyperactivation of mammalian target of rapamycin (mTOR) signaling pathway occurs after an epileptogenic insult and, its inhibition prevents the development of spontaneous seizures. We have recently demonstrated that mTOR's inhibition by rapamycin (started before seizure onset), permanently reduces the development of spontaneous absence seizures in WAG/Rij rats, an animal model of absence epilepsy; furthermore, mTOR phosphorylation was increased in adult WAG/Rij rats' cortex, but not other brain areas. However, it was not clear whether this hyperphosphorylation was a cause or a consequence of absence seizure. Here, we have addressed this issue by analyzing immunohistochemically: (1) the brain levels of total and phosphorylated mTOR in young (before seizures) and adult WAG/Rij rats; (2) the proliferation of hippocampal neuronal stem/progenitor cells assessed by BrdU analysis at different ages. WAG/Rij rats have higher levels of total mTOR in several brain areas than Wistar rats; phospho-mTOR staining is higher in young WAG/Rij rats than control and adult WAG/Rij rats. Finally, the age-related decline in hippocampal neural progenitor cell proliferation rate was slower in WAG/Rij than Wistar rats. Our results support a role for persistent mTOR activation and consequent change in hippocampal progenitor cell proliferation during the epileptogenic process leading to the development of absence seizures in WAG/Rij rats.

Taxonomic and phenotypic characterization of yeasts isolated from worldwide cold rock-associated habitats.

Yeast strains isolated from rock samples collected from worldwide cold regions were identified by sequence analysis of the D1/D2 domains of the 26S rDNA gene and the ITS region followed by molecular phylogeny. Over 77% of yeasts isolates were Basidiomycota. Cryptococcus (orders Filobasidiales and Tremellales) and Rhodotorula (order Cystobasidiales) were the most frequent genera. About 40% of yeast isolates belonged to undescribed species. Almost all isolates were psychrotolerant. Urease and esterase were the most widely extracellular enzymatic activity at 4 °C and 20 °C. None of the strains exhibited extracellular protease, DNAse, cellulase, chitinase, and laccase activity. The taxonomic and ecological significance of yeasts associated to worldwide cold rocky habitats is discussed.

Isolation and screening of black fungi as degraders of volatile aromatic hydrocarbons.

Black fungi reported as degraders of volatile aromatic compounds were isolated from hydrocarbon-polluted sites and indoor environments. Several of the species encountered are known opportunistic pathogens or are closely related to pathogenic species causing severe mycoses, among which are neurological infections in immunocompetent individuals. Given the scale of the problem of environmental pollution and the phylogenetic relation of aromate-degrading black fungi with pathogenic siblings, it is of great interest to select strains able to mineralize these substrates efficiently without any risk for public health. Fifty-six black strains were obtained from human-made environments rich in hydrocarbons (gasoline car tanks, washing machine soap dispensers) after enrichment with some phenolic intermediates of toluene and styrene fungal metabolism. Based on ITS sequencing identification, the majority of the obtained isolates were members of the genus Exophiala. Exophiala xenobiotica was found to be the dominant black yeast present in the car gasoline tanks. A higher biodiversity, with three Exophiala species, was found in soap dispensers of washing machines. Strains obtained were screened using a 2,6-dichlorophenol-indophenol (DCPIP) assay, optimized for black fungi, to assess their potential ability to degrade toluene. Seven out of twenty strains tested were able to use toluene as carbon source.

Antarctic epilithic lichens as niches for black meristematic fungi.

Sixteen epilithic lichen samples (13 species), collected from seven locations in Northern and Southern Victoria Land in Antarctica, were investigated for the presence of black fungi. Thirteen fungal strains isolated were studied by both morphological and molecular methods. Nuclear ribosomal 18S gene sequences were used together with the most similar published and unpublished sequences of fungi from other sources, to reconstruct an ML tree. Most of the studied fungi could be grouped together with described or still unnamed rock-inhabiting species in lichen dominated Antarctic cryptoendolithic communities. At the edge of life, epilithic lichens withdraw inside the airspaces of rocks to find conditions still compatible with life; this study provides evidence, for the first time, that the same microbes associated to epilithic thalli also have the same fate and chose endolithic life. These results support the concept of lichens being complex symbiotic systems, which offer attractive and sheltered habitats for other microbes.

Potential extinction of Antarctic endemic fungal species as a consequence of global warming.

Cryomyces spp. are fungi adapted to the harsh conditions of the McMurdo Dry Valleys in the Antarctic. The structure of their cell wall is one of the main factors for their uncommon ability to survive external stressors. The cells are, in fact, embedded in a thick and strongly melanised cell wall encrusted with black rigid plaques giving a supplementary protection and making them practically impregnable and refractory even to commercial enzymes including chitinases and glucanases. The Antarctic fungus Lecanicillium muscarium CCFEE 5003, able to produce an arsenal of lytic enzymes, including chitinases and glucanases, is known for its ability to degrade the cell walls of different food spoiling and opportunistic fungi as well as plant pathogenic Oomycota. Active cells of Cryomyces spp. were cultivated in dual culture with the mycoparasitic fungus both in liquid and solid media. Light microscope observations revealed that the cell walls of Cryomyces were heavily decayed. This resulted in the release of protoplasts. Hyphae penetration was evident with both scanning and transmission electron microscope observations. Due to its ecological amplitude (i.e. temperature growth range 0-28 °C), the parasitic fungus could easily expand its area of distribution as a consequence of global warming by invading new areas towards the interior of the continent. The establishment of interactions with organisms living at present in border ecosystems may lead to extinction of extremely specialized and poorly competitive entities.

Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures.

Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi -Coniosporium perforans, Exophiala jeanselmei - and of the extremophilic fungus -Friedmanniomyces endolithicus - were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum.

Resistance to UV-B induced DNA damage in extreme-tolerant cryptoendolithic Antarctic fungi: detection by PCR assays.

Cryptoendolithic Antarctic black fungi are adapted to the harshest terrestrial conditions as in the ice-free area of the McMurdo Dry Valleys. Recently, surviving space simulated conditions proves their bewildering extremotolerance. In order to investigate the potential DNA damage and their response after UV-B exposition, two strains of Antarctic cryptoendolithic black fungi, Cryomyces antarcticus CCFEE 534 and Cryomyces minteri CCFEE 5187, were irradiated at different UV-B doses. Since conventional methods cannot be applied to these organisms, the effect on the genome was assessed by RAPD and rDNA amplification PCR based assays; the results were compared with the responses of Saccharomyces pastorianus DBVPG 6283 treated with the same conditions. Results showed that template activity was drastically inhibited in S. pastorianus after irradiation. Dramatic changes in the RAPD profiles showed after 30 min of exposure while the rDNA amplification of SSU, LSU, and ITS portions failed after 30, 60, and 90 min of exposure respectively. No alteration was detected in the templates of the Antarctic strains where both RAPD profiles and rDNA PCR amplifications were unaffected even after 240 min of exposure. The electroferograms of the rDNA portions of Cryomyces strains were perfectly readable and conserved whilst the analyses revealed a marked alteration in S. pastorianus confirming the high resistance of the Antarctic strains to UV-B exposure.

Sample preparation and 2-DE procedure for protein expression profiling of black microcolonial fungi.

The ecology and stress adaptation of black rock inhabiting fungi in hot and cold extreme environments are not yet well understood. Two-dimensional gel electrophoresis (2-DE) is a promising tool to study the protein expression profiling and the metabolic status of microorganisms under stress conditions. The sample preparation has been shown to be the bottleneck for high resolution protein separation in 2-DE. For this purpose conditions must be optimized to obtain reliable and reproducible results. In addition, due to a multilayered and strongly melanized cell wall of black microcolonial fungi, special protocols for cell disruption and processing are required. In the present study, the protocol for protein extraction was established and optimized for the black yeast Exophiala jeanselmei MA 2853. The same protocol was successfully examined also for the meristematic fungus Coniosporium perforans MA 1299. Among the three procedures evaluated, trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation, and phenol extraction combined with methanol/ammonium acetate precipitation, the latter showed to be the best method for black yeasts and meristematic fungi. Penicillium chrysogenum was used as reference strain.