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.

Speleomycology of Air in Stopica Cave (Serbia).

Fungi can colonize organic matter present in subterranean sites and have a significant role as dwellers in different microniches of cave habitats. In order to analyze the content of airborne fungal propagules in different parts of "Stopica Cave," a touristic site in Serbia, air sampling was carried out in three seasons during 2020, prior to and during the onset of COVID-19 pandemic. Culturable mycobiota was identified using both microscopic techniques and ITS region/BenA gene barcoding, while multivariate analyses were employed to establish the link between fungal taxa and different environmental factors. The maximal measured fungal propagule concentrations were recorded during spring sampling which were based on fungal propagule concentration categories; the cave environment matches the category V. A total of 29 fungal isolates were identified, while Aspergillus, Cladosporium, Fusarium, Lecanicillium, Mucor, and Penicillium were the most diverse genera. According to the trophic mode, most of the isolated fungal species were pathotrophs (75.86%), but when regarding ecological guilds, the most dominant were undefined saprobes and animal pathogens (41.38% for each). Show caves are especially vulnerable to human impacts, and the fungal propagules' concentration within the caves could be good indices for the level of ecological disturbance.

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.

A Variety of Fungal Species on the Green Frogs' Skin (Pelophylax esculentus complex) in South Banat.

In the last several decades, amphibian populations have been declining worldwide. Many factors have been linked to global amphibian decline, including habitat destruction, pollution, introduced species, global environmental changes, and emerging infectious diseases. Recent studies of amphibian skin infections were mainly focused on the presence of chytridiomycosis, neglecting other members of the frogs' skin communities. The diversity pattern of fungal dwellers on the skin of green frogs (Pelophylax esculentus complex) was investigated. A total of 100 adults were sampled from three localities in South Banat (northern Serbia) over three consecutive years and detected fungal dwellers were identified using light microscopy and ITS and BenA gene sequencing. Structures belonging to fungi and fungus-like organisms including a variety of spores and different mycelia types were documented in the biofilm formed on amphibian skin, and are classified into 10 groups. In total, 42 fungal isolates were identified to species, section, or genus level. The difference in mycobiota composition between sampling points (localities and green frog taxa) was documented. The highest number of fungal structures and isolates was recorded on the hybrid taxon P. esculentus and locality Stevanove ravnice. Parental species showed a markedly lower diversity than the hybrid taxon and were more similar in diversity patterns and were placed in the same homogenous group. The locality Stevanove ravnice exhibited more pronounced differences in diversity pattern than the other two localities and was placed in a distinct and separate homogenous group. Among the fungal isolates, the highest isolation frequency was documented for Alternaria alternata, Aspergillus sp. sect. Nigri, Epicoccum nigrum, Fusarium proliferatum, and Trichoderma atroviride. Among the documented species, dematiaceous fungi, causative agents of chromomycosis in amphibians, were also recorded in this research with high isolation frequency. Also, some rare fungal species such as Quambalaria cyanescens and Pseudoteniolina globosa are documented for the first time in this research as microbial inhabitants of amphibian skin.

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.

Penicillium and Talaromyces Species as Postharvest Pathogens of Pear Fruit (Pyrus communis) in Serbia.

Pears are one of the oldest and the third most important fruit species grown in temperate regions. They are consumed because of their nutritional and health benefits, in fresh form or as various processed products. This article resolves the etiology of the Penicillium-like mold symptoms on pear fruits in Serbia. Samples of pear fruits with blue mold and other Penicillium-like mold symptoms were collected in Serbia from 2016 to 2019, from four storages. The recovered isolates were identified and characterized according to a polyphasic approach. Morphological and physiological analyses were performed on three media and five temperatures, respectively. Four loci (internal transcribed spacer, beta-tubulin, calmodulin, and DNA-dependent RNA polymerase II second largest subunit) were used for sequencing, genetic identification, and phylogenetic analyses. The results of the identification by conventional and molecular methods were in agreement, and they revealed that the obtained isolates belong to five species: Penicillium crustosum, P. expansum, P. italicum, Talaromyces minioluteus, and T. rugulosus. In a pathogenicity test, P. crustosum, P. expansum, T. minioluteus, and T. rugulosus produced decay on artificially inoculated pear fruits, and P. italicum induced tissue response lesions. The results of this study are the first reports of T. minioluteus and T. rugulosus as postharvest pear pathogens. Also, these are the first world records of T. minioluteus, T. rugulosus, and P. italicum on fruits of European pear. Furthermore, this is the first finding of P. crustosum, P. expansum, P. italicum, T. minioluteus, and T. rugulosus on pear fruit in Serbia.

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.

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.