Characterization of Culturable Mycobiome of Newly Excavated Ancient Wooden Vessels from the Archeological Site of Viminacium, Serbia.

Two ancient wooden vessels, specifically a monoxyle (1st century BCE to 1st century CE) and shipwreck (15th to 17th century CE), were excavated in a well-preserved state east of the confluence of the old Mlava and the Danube rivers (Serbia). The vessels were found in the ground that used to be river sediment and were temporarily stored within the semi-underground exhibition space of Mammoth Park. As part of the pre-conservation investigations, the primary aim of the research presented was to characterize the culturable mycobiomes of two excavated wooden artifacts so that appropriate conservation procedures for alleviating post-excavation fungal infestation could be formulated. Utilizing culture-based methods, a total of 32 fungi from 15 genera were identified, mainly Ascomycota and to a lesser extent Mucoromycota sensu stricto. Soft-rot Ascomycota of genus Penicillium, followed by Aspergillus and Cephalotrichum species, were the most diverse of the isolated fungi. Out of a total of 38 isolates, screened on 7 biodegradation plate assays, 32 (84.21%) demonstrated at least one degradative property. Penicillium solitum had the highest deterioration potential, with a positive reaction in 5 separate plate assays. The obtained results further broaden the limited knowledge on the peculiarities of post-excavation soft-rot decay of archaeological wood and indicate the biochemical mechanisms at the root of post-excavation fungal deterioration.

Insights into Endophytic and Rhizospheric Bacteria of Five Sugar Beet Hybrids in Terms of Their Diversity, Plant-Growth Promoting, and Biocontrol Properties.

Sugar beet is the most important crop for sugar production in temperate zones. The plant microbiome is considered an important factor in crop productivity and health. Here, we investigated the bacterial diversity of seeds, roots, and rhizosphere of five sugar beet hybrids named Eduarda (ED), Koala (KO), Tibor (T), Tajfun (TF), and Cercospora-resistant (C). A culture-independent next-generation sequencing approach was used for the further investigation of seed-borne endophytes. Hybrid-associated bacteria were evaluated for their plant growth-promoting (PGP) characteristics, antagonistic activity towards Cercospora beticola and several Fusarium strains in dual culture assays, and drought and salinity tolerance. High-throughput sequencing revealed that the Proteobacteria phylum was most dominant in the seeds of all hybrids, followed by Cyanobacteria and Actinobacteriota. The predominant genus in all hybrids was Pantoea, followed by Pseudomonas, Acinetobacter, Chalicogloea, Corynebacterium, Enterobacter, Enterococcus, Glutamicibacter, Kosakonia, and Marinilactibacillus. Unique genera in the hybrids were Pleurocapsa and Arthrobacter (T), Klebsiella (TF), Apibacter (ED), and Alloscardovia (KO). The genera that were most represented in one hybrid were Weissella and Staphylococcus (TF); Streptococcus (T); Gardnerella, Prevotella, and Rothia (KO); and Gilliamella, Lactobacillus, and Snodgrassella (ED). Thirty-two bacteria out of 156 isolates from the rhizosphere, roots, and seeds were selected with respect to various plant growth-promoting activities in vitro, i.e., nitrogen fixation, phosphate solubilization, siderophore production, indole-3-acetic acid production, 1-aminocyclopropane-1-carboxylic acid deaminase activity, hydrogen cyanide production, exoenzymatic activity (amylase, protease, lipase, cellulase, xylanase, mannanases, gelatinase, and pectinase), mitigation of environmental stresses, and antifungal activity. Mixta theicola KO3-44, Providencia vermicola ED3-10, Curtobacterium pusillum ED2-6, and Bacillus subtilis KO3-18 had the highest potential to promote plant growth due to their multiple abilities (nitrogen fixation, phosphate solubilization, production of siderophores, and IAA). The best antagonistic activity towards phytopathogenic fungi was found for Bacillus velezensis C3-19, Paenibacillus polymyxa C3-36 and Bacillus halotolerans C3-16/2.1. Only four isolates B. velezensis T2-23, B. subtilis T3-4, B. velezensis ED2-2, and Bacillus halotolerans C3-16/2.1 all showed enzymatic activity, with the exception of xylanase production. B. halotolerans C3-16/2.1 exhibited the greatest tolerance to salinity, while two B. subtilis strains (C3-62 and TF2-1) grew successfully at the maximum concentration of PEG. The current study demonstrates that sugar beet-associated bacteria have a wide range of beneficial traits and are therefore highly promising for the formulation of biological control and PGP agents.

Bacteriobiota of the Cave Church of Sts. Peter and Paul in Serbia-Culturable and Non-Culturable Communities' Assessment in the Bioconservation Potential of a Peculiar Fresco Painting.

The principal aim of this study was to determine bacterial diversity within the Cave Church of Sts. Peter and Paul, via culturable and non-culturable approaches, and elucidate the antifungal potential of autochthonous antagonistic bacterial isolates against biodeteriogenic fungi. Furthermore, whole-genome sequencing of selected bacterial antagonists and the analysis of genes included in the synthesis of secondary metabolites were performed. With the highest RA values, determined in metabarcoding analysis, phyla Actinobacteriota (12.08-54.00%) and Proteobacteria (25.34-44.97%) dominated most of the samples. A total of 44 different species, out of 96 obtained isolates, were determined as part of the culturable bacteriobiota, with the predominance of species from the genus Bacillus. Bacillus simplex was the only isolated species simultaneously present in all investigated substrata within the church. The best antagonistic activity against 10 biodeteriogenic fungi was documented for Streptomyces anulatus, followed by Bacillus altitudinis, Chryseobacterium viscerum, and Streptomyces sp. with their highest PGI% values ranging of from 55.9% to 80.9%. These promising results indicate that characterized bacteria are excellent candidates for developing biocontrol strategies for suppressing deteriogenic fungi responsible for the deterioration of investigated fresco painting. Finally, isolate 11-11MM, characterized as Streptomyces sp., represents a new species for science prompting the need for further study.

An Insight into an Olive Scab on the "Istrska Belica" Variety: Host-Pathogen Interactions and Phyllosphere Mycobiome.

The olive tree is one of the most important agricultural plants, affected by several pests and diseases that cause a severe decline in health status leading to crop losses. Olive leaf spot disease caused by the fungus Venturia oleaginea can result in complete tree defoliation and consequently lower yield. The aim of the study was to obtain new knowledge related to plant-pathogen interaction, reveal mechanisms of plant defense against the pathogen, and characterize fungal phyllosphere communities on infected and symptomless leaves that could contribute to the development of new plant breeding strategies and identification of novel biocontrol agents. The highly susceptible olive variety "Istrska Belica"' was selected for a detailed evaluation. Microscopy analyses led to the observation of raphides in the mesophyll and parenchyma cells of infected leaves and gave new insight into the complex V. oleaginea pathogenesis. Culturable and total phyllosphere mycobiota, obtained via metabarcoding approach, highlighted Didymella, Aureobasidium, Cladosporium, and Alternaria species as overlapping between infected and symptomless leaves. Only Venturia and Erythrobasidium in infected and Cladosporium in symptomless samples with higher abundance showed statistically significant differences. Based on the ecological role of identified taxa, it can be suggested that Cladosporium species might have potential antagonistic effects on V. oleaginea.

Mycobiome Diversity of the Cave Church of Sts. Peter and Paul in Serbia-Risk Assessment Implication for the Conservation of Rare Cavern Habitat Housing a Peculiar Fresco Painting.

The mycobiome of the cave Church of Sts. Peter and Paul, housing the peculiar fresco painting of "The Bald-headed Jesus", was analyzed via culture-dependent and -independent methods. Salt efflorescence, colored patinas, and biofilm, as well as biopitting, discolorations, and fruiting bodies of wood-decay fungi were observed on surfaces within the church. Microscopic analyses showed an abundance of fungal structures, i.e., conidiophores, conidia, chlamydospores, and ascospores. The estimated values of the contamination classified all surfaces as the "Danger zone". A total of 24 fungi from 17 genera were determined as part of the culturable mycobiome, with a dominance of Ascomycota of genera Penicillium. Biodegradative profiles analyzed via plate assays demonstrated positive reactions for 16 isolates: most commonly acid production (8), followed by pigment production and ligninolytic activity (6), protein degradation (5), cellulolytic activity (3) and carbonate dissolution (2). Metabarcoding analysis showed a dominance of Ascomycota in all samples (79.9−99.7%), with high relative abundance documented for Hypoxylon fuscopurpureum on the iconostasis and unclassified Mycosphaerellaceae family within order Capnodiales on fresco and stone, as well as moderate relative abundance for unclassified Dothideomycetes, Botryolepraria lesdainii, Verrucaria sp. and Cladosporium sp. on stone walls. The used set of integrative methods pointed out species of genus Neodevriesia and H. fuscopurpureum as the main deteriogenic agents of fresco and iconostasis surfaces, respectively.

The microbiome of bat guano: for what is this knowledge important?

Bats as flying mammals are potent vectors and natural reservoir hosts for many infectious viruses, bacteria, and fungi, also detected in their excreta such as guano. Accelerated deforestation, urbanization, and anthropization hastily lead to overpopulation of the bats in urban areas allowing easy interaction with other animals, expansion, and emergence of new zoonotic disease outbreaks potentially harmful to humans. Therefore, getting new insights in the microbiome of bat guano from different places represents an imperative for the future. Furthermore, the use of novel high-throughput sequencing technologies allows better insight in guano microbiome and potentially indicated that some species could be typical guano-dwelling members. Bats are well known as a natural reservoir of many zoonotic viruses such as Ebola, Nipah, Marburg, lyssaviruses, rabies, henipaviruses, and many coronaviruses which caused a high number of outbreaks including ongoing COVID-19 pandemic. Additionally, many bacterial and fungal pathogens were identified as common guano residents. Thus, the presence of multi-drug-resistant bacteria as environmental reservoirs of extended spectrum ß-lactamases and carbapenemase-producing strains has been confirmed. Bat guano is the most suitable substrate for fungal reproduction and dissemination, including pathogenic yeasts and keratinophilic and dimorphic human pathogenic fungi known as notorious causative agents of severe endemic mycoses like histoplasmosis and fatal cryptococcosis, especially deadly in immunocompromised individuals. This review provides an overview of bat guano microbiota diversity and the significance of autochthonous and pathogenic taxa for humans and the environment, highlighting better understanding in preventing emerging diseases. KEY POINTS: Bat guano as reservoir and source for spreading of autochthonous and pathogenic microbiota Bat guano vs. novel zoonotic disease outbreaks Destruction of bat natural habitats urgently demands increased human awareness.

Phyllosphere Fungal Communities of Plum and Antifungal Activity of Indigenous Phenazine-Producing Pseudomonas synxantha Against Monilinia laxa.

European plum (Prunus domestica L.) is a significant commercial crop in Serbia in terms of total fruit production, and is traditionally processed into slivovitz brandy. The brown rot disease caused by Monilinia laxa drastically reduces plum yield almost every year. Fungal communities associated with leaves and fruits of four local Serbian plum cultivars (Pozegaca, Ranka, Cacanska Lepotica and Cacanska Rodna) were investigated in two phenological stages during early (May) and late (July) fruit maturation. Alpha diversity indices showed that fungal communities were heterogeneous and Beta diversity indicated that autochthonous fungal communities depended upon seasonal changes and the cultivars themselves. The phylum Ascomycota was the most abundant in all samples, with relative abundance (RA) between 46% in the Pozegaca cultivar (May) and 89% in the Lepotica cultivar (July). The most abundant genus for all plum cultivars in May was Aureobasidium, with RA from 19.27 to 33.69%, followed by Cryptococcus, with 4.8 to 48.80%. In July, besides Cryptococcus, different genera (Metschnikowia, Fusarium, and Hanseniaspora) were dominant on particular cultivars. Among all cultivable fungi, molecular identification of eleven M. laxa isolates from four plum cultivars was performed simultaneously. Bacterial isolates from the plum phyllosphere were tested for their potential antifungal activity against indigenous M. laxa isolates. The most potent antagonist P4/16_1, which significantly reduced mycelial growth of M. laxa, was identified as Pseudomonas synxantha. Further characterization of P4/16_1 revealed the production of volatile organic compounds and phenazine-1-carboxylic acid (PCA). Crude benzene extract of PCA exhibited 57-63% mycelial growth inhibition of M. laxa. LC/MS analysis of the crude extract confirmed the presence of phenazine derivatives amongst other compounds. Scanning electron microscopy revealed morpho-physiological changes in the hyphae of M. laxa isolates caused by the cell culture and the P. synxantha P4/16_1 crude benzene extract. This is the first report of antagonistic activity of P. synxantha against M. laxa induced by diffusible and volatile antifungal compounds, and it appears to be a promising candidate for further investigation for potential use as a biocontrol agent against brown rot-causing fungi.

Phenol-based millipede defence: antimicrobial activity of secretions from the Balkan endemic millipede Apfelbeckia insculpta (L. Koch, 1867) (Diplopoda: Callipodida).

Millipedes use an array of chemical compounds to defend themselves from predator attack. These chemical substances can have additional roles, i.e. defence against various pathogens. We evaluated the efficacy of the defensive secretion of Apfelbeckia insculpta (L. Koch, 1867) against bacteria, yeasts, and filamentous fungi. The tested secretion consisted of two compounds, p-cresol and phenol, and showed antibacterial, antibiofilm, and antifungal potential against all selected microorganisms. The most sensitive bacterium in our study was Pseudomonas aeruginosa, while the tested defensive secretion manifested the lowest activity against Escherichia coli. The defensive secretion of A. insculpta also showed an ability, albeit mild, to suppress biofilm formation by P. aeruginosa. Among the tested yeasts, Candida albicans and C. krusei were the most susceptible and most resistant species, respectively. Finally, the concentration of extracts obtained from the tested defensive secretion needed to achieve an antifungal effect was lowest in the case of Cladosporium cladosporioides. Fusarium verticillioides and Penicillium rubens were the micromycetes most resistant to the tested secretion. Our results indicate that antibacterial activity of the defensive secretion of A. insculpta is similar to or slightly weaker than that of streptomycin, while comparison with antimycotics showed that the tested millipede secretion has stronger activity than fluconazole, but weaker activity than nystatin and ketoconazole. The present study corroborates previous findings indicating that the defensive secretions of millipedes can have different roles apart from antipredator protection and are effective against pathogenic microorganisms.

In vitro biodegradation potential of airborne Aspergilli and Penicillia.

The study addresses in vitro degradation potential of airborne Aspergillus and Penicillium/Talarmyces species originating from cultural heritage conservation premises. A series of rapid, cost effective biodegradation assays were performed to assess production of extracellular pigments, acids, and enzymes. Most of the isolates have demonstrated positive growth in at least one of the preformed tests. Strongest overall degradation potential was demonstrated for Penicillium brevicompactum, P. glabrum, and Talaromyces sayulitensis while Aspergillus domesticus, A. penicillioides, A. pseudoglaucus, and A. ruber did not exhibit positive reaction in any of the employed assays. Majority of isolates exhibited proteolytic and cellulolytic activity while carbonate dissolution was observed for only five tested fungi. Highest alteration of pH value in liquid media was documented for T. sayulitensis while A. niger and P. expansum exhibited strongest acid production on CREA. Certain isolates, mostly Penicillium species, displayed production of extracellular pigments. The results imply that many of the tested fungi have significant biodegradation capacity, indicating their potential to inflict structural and esthetic alterations on cultural heritage objects.

Multifaceted activity of millipede secretions: Antioxidant, antineurodegenerative, and anti-Fusarium effects of the defensive secretions of Pachyiulus hungaricus (Karsch, 1881) and Megaphyllum unilineatum (C. L. Koch, 1838) (Diplopoda: Julida).

Members of the millipede order Julida rely on dominantly quinonic defensive secretions with several minor, non-quinonic components. The free radical-scavenging activities of ethanol, methanol, hexane, and dichloromethane extracts of defensive secretions emitted by Pachyiulus hungaricus (Karsch, 1881) and Megaphyllum unilineatum (C. L. Koch, 1838) were investigated using the ABTS, DPPH, and total reducing power (TRP) tests. The obtained extracts were also tested for inhibition of acetylcholinesterase and tyrosinase activity. Finally, the antifungal potential of both julid extracts was evaluated against seven Fusarium species. Secretions of both species showed activity against free radicals, acetylcholinesterase, tyrosinase, and all of the selected fungal species. The secretions of P. hungaricus exhibited a more potent antioxidative effect than did those of M. unilineatum, while there were no significant differences of antiacetylcholinesterase activity between the tested extracts. Only the hexane extract of M. unilineatum showed an effect on tyrosinase activity stronger than that of P. hungaricus. Fusarium sporotrichioides, F. graminearum, and F. verticillioides were the fungi most resistant to secretions of both julids. The Fusarium species most susceptible to the secretion of P. hungaricus was F. avenaceum, while the concentrations of M. unilienatum extracts needed to inhibit and completely suppress fungal growth were lowest in the case of their action on F. lateritium. Our data support previous findings that julid defensive secretions possess an antimicrobial potential and reveal their antioxidative and antineurodegenrative properties. Bearing in mind the chemical complexity of the tested defensive secretions, we presume that they can also exhibit other biological activities.

ATP bioluminescence method: tool for rapid screening of organic and microbial contaminants on deteriorated mural paintings.

The extent of the microbial contamination of the seventeenth-century wall paintings in the nave of the old Church of the Holy Ascension (Veliki Krcimir, Serbia) was evaluated via newly implemented ATP bioluminescence method, and traditional cultivation-based method, utilising commercially available dip slides. To assess the validity of ATP, as a biomarker for rapid detection of mural surface contamination, obtained zones of cleanliness values, in range from 1.0 to 5.3, were compared to documented total microbial counts, ranging between seven and 247 CFU/cm2. Small coefficients of determination, 0.0106-0.0385, suggest poor correlation between microbial counts and surface ATP levels; however, zones of cleanliness values are of great help in determining the high points of contamination, aka 'hotspots', which should be given special attention during sampling and investigation using other methods. In addition, various aspects of the possible implementation of the ATP bioluminescence method in an integrated system of wall painting conservation are discussed.

Frankincense and myrrh essential oils and burn incense fume against micro-inhabitants of sacral ambients. Wisdom of the ancients?

ETHNOPHARMACOLOGICAL RELEVANCE: Essential oils obtained from resins of Boswellia carteri Birdw. and Commiphora myrrha (Nees) Engl., commonly known as frankincense and true myrrh respectively, have been used extensively since 2800 BCE for the treatment of skin sores, wounds, teeth, inflammation, and urinary tract diseases in traditional medicine; for preparation of mummification balms and unguents; and also as incense and perfumes. Since ancient times, burning of frankincense and myrrh in places of worship for spiritual purposes and contemplation (a ubiquitous practice across various religions) had hygienic functions, to refine the smell and reduce contagion by purifying the indoor air. AIM OF THE STUDY: The general purpose of the study was to assess the in vitro antimicrobial potential of the liquid and vapour phases of B. carteri and C. myrrha essential oils and burn incense, as well as to test the effectiveness of their in situ application to cleanse microbially-contaminated air within the ambient of an investigated 17th-century church. MATERIALS AND METHODS: The chemical composition of B. carteri and C. myrrha essential oils, obtained by hydrodistillation of frankincense and true myrrh oleo gum resins was determined using GC/MS, and antimicrobial properties of their liquid and vapour phases were assessed by the broth microdilution and microatmosphere diffusion methods. Chemical analysis of burn incense fume obtained using bottle gas washing with dichloromethane as a solvent was performed by GC/MS, while its antimicrobial activity was evaluated using a modified microatmosphere diffusion method to evaluate germination inhibition for fungi and CFU count reduction for bacteria. The in situ antimicrobial activity of B. carteri burn incense and essential oil vapour phase was assessed in the sealed nave and diaconicon of the church, respectively. RESULTS: The dominant compounds of B. carteri EO were α-pinene (38.41%) and myrcene (15.21%), while C. myrrha EO was characterized by high content of furanoeudesma-1,3-diene (17.65%), followed by curzerene (12.97%), ß-elemene (12.70%), and germacrene B (12.15%). Burn incense fume and soot had α-pinene (68.6%) and incensole (28.6%) as the most dominant compounds, respectively. In vitro antimicrobial assays demonstrated high bacterial and fungal sensitivity to the liquid and vapour phases of EOs, and burn incense fume. In situ application of B. carteri EO vapour and incense fume resulted in reduction of air-borne viable microbial counts by up to 45.39 ±â€¯2.83% for fungi and 67.56 ±â€¯3.12% for bacteria (EO); and by up to 80.43 ±â€¯2.07% for fungi and 91.43 ±â€¯1.26% for bacteria (incense fume). CONCLUSIONS: The antimicrobial properties of essential oil derived from frankincense, a compound with well-known traditional use, showed that it possesses a clear potential as a natural antimicrobial agent. Moreover, the results suggest possible application of B. carteri EO vapour and incense fume as occasional air purifiers in sacral ambients, apart from daily church rituals.

Biodegradative potential of fungal isolates from sacral ambient: In vitro study as risk assessment implication for the conservation of wall paintings.

The principal purpose of the study was to evaluate in vitro the potential ability of fungal isolates obtained from the painted layer of frescoes and surrounding air to induce symptoms of fresco deterioration, associated with their growth and metabolism, so that the risk of such deterioration can be precisely assessed and appropriate conservation treatments formulated. Biodegradative properties of the tested microfungi were qualitatively characterized through the use of a set of special agar plates: CaCO3 glucose agar (calcite dissolution), casein nutrient agar (casein hydrolysis), Czapek-Dox minimal medium (pigment secretion); and Czapek-Dox minimal broth (acid and alkali production). Most of the tested isolates (71.05%) demonstrated at least one of the degradative properties, with Penicillium bilaiae as the most potent, since it tested positive in all four. The remaining isolates (28.95%) showed no deterioration capabilities and were hence considered unlikely to partake in the complex process of fungal deterioration of murals via the tested mechanisms. The obtained results clearly indicate that utilization of fast and simple plate assays can provide insight into the biodegradative potential of deteriogenic fungi and allow for their separation from allochthonous transients, a prerequisite for precise assessment of the amount of risk posed by a thriving mycobiota to mural paintings.

Seasonal diversity of biodeteriogenic, pathogenic, and toxigenic constituents of airborne mycobiota in a sacral environment.

The main purpose of this study was to isolate airborne fungi and assess seasonal variations in air contamination with their particulates by determining the levels of their propagules in the nave and exonarthex of a church. We also monitored indoor microclimate as a determining factor for fungal proliferation on wall paintings, spore release, and transmission through the air. The temperature and relative humidity of the nave favoured fungal growth. A total of 33 fungi were isolated, mainly of the phylum Ascomycota, and to the lesser extent of the phyla Zygomycota and Basidiomycota. The most common were the fungi of the genera Penicillium and Aspergillus (23.55 % and 20.58 %, respectively). Sørensen's quotient of similarity (0.37) suggests moderate species overlap and constant exchange of fungal propagules between the nave and exonarthex. The autumn had the highest diversity, with 17 documented taxa, followed by the summer and the winter. The spring had only eight taxa. Quantitative analysis of the airborne mycobiota in the nave (430±84.85 to 1880±106.07 CFU m-3) and exonarthex (715±59.62 to 2295±91.92 CFU m-3) showed very high contamination throughout the year, with values exceeding the maximum permissible concentrations by most standards. Many of the fungi determined in this study are known for their biodeteriogenic, toxigenic, and allergenic properties, and are a threat not only to occasional visitors and staff, but also to valuable works of art decorating nave walls.

Biogenesis of secondary mycogenic minerals related to wall paintings deterioration process.

Present study addresses potential of fungal strains, isolated from deteriorated mural paintings and surrounding air environment of the Church of the Holy Ascension in Veliki Krcimir (Serbia), to precipitate mycogenic minerals, when cultivated on agarized B4 medium. Utilizing culture-based isolation methods, 38 filamentous fungi were obtained in total, 23 from mural paintings and 15 from air, respectively, mainly ascomycetes, while Bjerkandera adusta and Thanatephorus cucumeris were only basidiomycetes. A total of 31 of 38 fungal isolates, more than 80%, were able to form minerals of different morphologies and variable size, determined via SEM-EDS and XRPD, to be either calcite or calcite and weddellite association. Among screened fungi, all Penicillium, Chaetomium and Cladosporium isolates, as well as most of the Aspergillus isolates (8/11) precipitated minerals, whereas cultures of Bionectria, Bjerkandera, and Seimatosporium isolates lacked any observable crystal forms. With the exception of two Alternaria alternata strains, no apparent disparity in potential to precipitate minerals in general, or form particular crystal phase was documented among the air and mural paintings isolates. Possible mechanisms of fungal mineralization of calcite and weddellite are further proposed. In addition to providing experimental evidence for fungal induced precipitation of oxalate and carbonate minerals, presented data suggest that fungal activity could be an important factor in a weathering process affecting cultural heritage exhibited and stored in inadequate conditions. Implementation of B4 plate assay for screening of mineralization potential of the isolated fungi could be used to assess biodegradative risk mycobiota pose to the mural paintings, so appropriate conservation measures may be utilized.

Fungal-Induced Deterioration of Mural Paintings: In Situ and Mock-Model Microscopy Analyses.

Fungal deterioration of frescoes was studied in situ on a selected Serbian church, and on a laboratory model, utilizing standard and newly implemented microscopy techniques. Scanning electron microscopy (SEM) with energy-dispersive X-ray confirmed the limestone components of the plaster. Pigments used were identified as carbon black, green earth, iron oxide, ocher, and an ocher/cinnabar mixture. In situ microscopy, applied via a portable microscope ShuttlePix P-400R, proved very useful for detection of invisible micro-impairments and hidden, symptomless, microbial growth. SEM and optical microscopy established that observed deterioration symptoms, predominantly discoloration and pulverization of painted layers, were due to bacterial filaments and fungal hyphal penetration, and formation of a wide range of fungal structures (i.e., melanized hyphae, chlamydospores, microcolonial clusters, Cladosporium-like conidia, and Chaetomium perithecia and ascospores). The all year-round monitoring of spontaneous and induced fungal colonization of a "mock painting" in controlled laboratory conditions confirmed the decisive role of humidity level (70.18±6.91% RH) in efficient colonization of painted surfaces, as well as demonstrated increased bioreceptivity of painted surfaces to fungal colonization when plant-based adhesives (ilinocopie, murdent), compared with organic adhesives of animal origin (bone glue, egg white), are used for pigment sizing.

Aspergilli Response to Benzalkonium Chloride and Novel-Synthesized Fullerenol/Benzalkonium Chloride Nanocomposite.

A comprehensive comparative analysis of antifungal potential of benzalkonium chloride and newly synthesized fullerenol/benzalkonium chloride nanocomposite was conducted to assess the possible impact of carbon-based nanocarrier on antimicrobial properties of the commonly used biocide. Physical characterization of synthesized nanocomposite showed zeta potential of +37.4 mV and inhomogeneous particles size distribution, with nanocomposite particles' dimensions within 30-143 nm and maximum number of particles at 44 nm. The effect of pure and fullerenol nanocarrier-bound biocide was evaluated in eight Aspergillus species. In mycelial growth assay, nanocomposite was more potent, as fungicidal effect of 1.04/0.6 µg mL(-1) was obtained in all but one of the isolates (A. niger), while proportional concentration of pure biocide (0.6 µg mL(-1)) completely inhibited mycelial growth of only three Aspergillus species. However, conidia appear to be less susceptible to nanocomposite treatment, as lower fungistatic (MIC) and fungicidal (MFC) concentrations were obtained with biocide alone (MIC in range from 0.03 to 0.15 µg mL(-1) and MFC from 0.075 to 0.45 µg mL(-1)). To a different degree, both substances stimulated aflatoxin B1 production and inhibited ochratoxin A synthesis. Very low mycelium biomass yield, in range from 1.0 to 3.0 mg dry weight, was documented in both biocide and nanocomposite enriched medium.