Diversity and role of micromycetes in wall painting biodeterioration process: Church of the Holy Ascension in Veliki Krčimir

Analiziran je diverzitet i sezonska dinamika mikromiceta na zidnim slikama i u vazduhu istraživane crkve. Identifikovana je mikobiota od 46 taksona mikromiceta, sa dominacijom Aspergillus, Penicillium i Cladosporium vrsta. Najveći diverzitet je zabeležen tokom leta, dok je najveća brojnost propagula po jedinici površine (“fungalni otisak”) konstatovana u zimu. Aeromikobiota je okarakterisana sa 33 taksona gljiva. Kontaminacija vazduha propagulama gljiva tokom godine višestruko je prevazilazila standarde za zatvorene prostore. Mikroskopska analiza biofilma pokazala je da su reproduktivne strukture Cladosporium sp. i Chaetomium sp. u kontaktu bojenog sloja i maltera glavni biotski faktor deterioracije. Metodom ATP bioluminiscenije, 75% površina zidnih slika je okarakterisano kao “Zona opasnosti”. Monitoringom indukovane i spontane kolonizacije na modelu zidne slike zaključeno je da su lihenizovane i mikrokolonijalne gljive uzrok fenomena “biopitting”. Testirani izolati su demonstrirali veliki potencijal deterioracije zidnih slika u eksperimentima in vitro. BAC i novosintetisani BAC/FNP nanokompozit pokazali su dobru antifungalnu aktivnost, kao i uticaj na povećanje produkcije aflatoksina B1 i smanjenje produkcije ohratoksina A. Etarsko ulje tamjana imalo je jači antifungalni efekat u odnosu na etarsko ulje smirne, ali slabiji u poređenju sa smešom ulja. Dim tamjana poseduje inhibitorno dejstvo na germinaciju konidija, mehaničkim i hemijskim dejstvom voštanog sloja deponovanog iz dima, in vitro. Tretman vazduha crkve dimom tamjana redukovao je nivo kontaminacije vazduha fungalnim propagulama za približno 80%. Preko predloženog metodološkog protokola, data je mogućnost implementacije rezultata ovog istraživanja u praksu konzervacije i restauracije kulturnih dobara.Diversity and seasonal dynamics of micromycetes from wall paintings and surrounding air of investigated church was analyzed. A total of 46 fungal taxa was documented, with dominance of Aspergillus, Penicillium and Cladosporium species. The greatest species diversity was recorded in the summer, while the highest abundance of fungal propagules per unit area (“fungal print”) was observed in the winter. Air mycobiota was characterized by 33 fungal taxa. Fungal air contamination, throughout the year, exceeded permisable levels for indoor environment. Microscopic biofilm analyzes revealed that reproductive structures of Cladosporium sp. and Chaetomium sp. are the main biodeteriogens. ATP bioluminescence method showed that 75% of the surfaces are deemed as “Danger zone”. Monitoring of induced and spontaneous colonization of mock-model established lichenized and micro-colonial fungi as the main causative agents of “biopitting” phenomenon. Tested isolates demonstrated pronounced deterioration capabilities in experiments in vitro. BAC and newlysynthesized BAC/FNP nanocomposite had very strong antifungal activity, stimulated aflatoxin B1 production, and inhibited ochratoxin A production. Frankincense essential oil showed stronger antifungal property compared to the oil obtained from myrrh, but weaker than the oil mixture. Burn incense had inhibitory effect on conidia germination via mechanical and chemical activity of thin waxy layer deposited from fume, in vitro. Treatment of air within church, with burn incense, reduced the level of fungal air contamination by approximatly 80%. Through the proposed methodological protocol, the possibility of implementing results into the practice of conservation and restoration of cultural heritage is given

Preserving the Danube Limes in Serbia: A Review on the Biodeterioration of Trajan’s Bridge

One of the most significant monuments of the Roman Danube Limes is Trajan's Bridge, built in the period from 103 to 105 AD. The remains of the pillars on the Serbian bank of the Danube were partially restored four decades ago. Today, the pillar that is closest to the river, which has not undergone conservation, is subject to different types of deterioration. During the comparison between the present condition of the pillar and those recorded in photos over the last six decades, we can estimate that its level of material loss has not overly changed. However, the difference in biological growth is visible. The primary aim of this study was to record the degree of the infestation and endangerment of the monument and check for possible risks for future mortar deterioration where mortar is in direct contact with severely infested bricks. Moreover, a characterisation of the biodeteriogens was performed. Special emphasis was given to fungi as the main agents of deterioration. A precise assessment of the level of risk they pose to this monument was made as a basis for the formulation and implementation of appropriate conservation treatments. Severe macrofouling by epilithic lichenised fungi and mosses was documented. Even in areas where pillars lack visible infestation, thriving microbial communities characterized by the presence of various fungal structures, as well as structures of trichal Cyanobacteria, and Chlorophyta were recorded. Similarities between communities documented on brick, mortar, and stone surfaces estimated via Sørensen’s quotient of similarity, were high, with the highest similarity documented between mortar and stone

Supplementary data for the article: Janakiev, T.; Dimkić, I.; Unković, N.; Ljaljević Grbić, M.; Opsenica, D.; Gašić, U.; Stanković, S.; Berić, T. Phyllosphere Fungal Communities of Plum and Antifungal Activity of Indigenous Phenazine-Producing Pseudomonas Synxantha Against Monilinia Laxa. Frontiers in Microbiology 2019, 10. https://doi.org/10.3389/fmicb.2019.02287

Supplementary material for: [https://doi.org/10.3389/fmicb.2019.02287]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3729

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 (Požegača, Ranka, Čačanska Lepotica and Čačanska 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 Požegača 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.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3730

Assessment of growth-promoting properties of Pseudomonas spp. on soybeans under field conditions

The soybean is recognized worldwide as one of the most important crops due to its protein- and oil-rich seeds. Several beneficial bacterial strains, including those of the genus Pseudomonas, are known to increase plant yield and disease tolerance. The aim of this study was to test the potential of 15 strains of Pseudomonas spp. to promote soybean growth under field conditions. Strains were identified based on 16S rRNA and gyrB sequences, and strains belonging to risk group 1 were further analyzed. Selected strains were tested for plant growth-promoting (PGP) properties, biofilm formation and in vitro antimicrobial activity against various fungal pathogens. PGP activity indicators of the selected strains were evaluated on soybean plants grown in fertilizer-free soil and in soil treated with 70% and 100% fertilizers. Three Pseudomonas spp. strains, P. putida VB131A, P. fulva VB134B and P. rhodesiae VB143S, were selected as PGP candidates. All three strains showed the ability to produce IAA and solubilize phosphorus, while only P. rhodesiae VB143S showed strong lipase and protease activity. Only P. fulva VB134B showed the ability to produce biofilm and hydrogen cyanide. All three strains showed in vitro antifungal activity against Alternaria infectoria. P. fulva VB134B and P. rhodesiae VB143S showed antifungal activity against Alternaria alternata and P. putida VB131A and P. rhodesiae VB143S against Epicoccum nigrum. Only P. fulva VB134B showed activity against Monilinia laxa. Statistically significant differences in plant height were not observed regardless of the strain or soil treatment used. In terms of first pod height, the P. fulva VB134B treatment was most effective when applied to soybeans grown in 100% fertilized soil. In contrast, plants treated with P. rhodesiae VB143S and grown in 100% fertilized soil had the highest number of branches and pods per plant

Fungal Deterioration of Cultural Heritage Objects

Significant percent of world cultural heritage artifacts is threatened by fungal infestation. Fungi can deteriorate different substrates via various physical and chemical mechanisms. Hyphal growth and penetration into the substrate can cause symptoms like discoloration, biopitting, cracking, exfoliation and patina formation. On the other hand, chemical mechanisms include acid secretion, release of extracellular enzymes, pigment production, oxidation/reduction reactions and secondary mycogenic minerals formation. These processes can lead to serious, both esthetic and structural, alterations which may be irreversible and could permanently impair artworks. Proper isolation and identification of autochthonous isolates, as well as employment of different microscopic techniques and in vitro biodegradation tests are pivotal in understanding complex biodeterioration mechanisms caused by microorganisms, including fungal deteriogens. Biodeterioration and biodegradation studies require multidisciplinary approach and close collaboration of microbiologists, chemists, geologists and different personnel responsible for the safeguarding of cultural heritage monuments and artifacts, especially restorers and conservators

Supplementary material for the article: Ilić, B.; Dimkić, I.; Unković, N.; Grbić, M. L.; Vukojević, J.; Vujisić, L.; Tešević, V.; Stanković, S.; Makarov, S.; Lučić, L. Millipedes vs. Pathogens: Defensive Secretions of Some Julids (Diplopoda: Julida) as Potential Antimicrobial Agents. Journal of Applied Entomology 2018, 142 (8), 775–791. https://doi.org/10.1111/jen.12526

Supplementary material for: [https://doi.org/10.1111/jen.12526]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2198

Mikroorganizmi u sastavu biofilma s različitih supstrata staklenika Botaničke bašte “Jevremovac”

Diversity of subaerial biofilm forming cyanobacteria, algae and fungi was investigated on 10 different substrata from greenhouse of Botanical Garden “Jevremovac”. Out of 37 documented taxa, 16 cyanobacterial and 10 algal taxa were identified. Remaining 11 taxa belong to the Kingdom of Fungi. The highest diversity of biofilm forming microorganisms, a total of 24 taxa, was detected on the corroded metal surface, while significantly lower number of taxa was recorded on other examined substrata. Cyanobacterium Porphyrosiphon sp., diatom Achnanthes sp. and green algae Chlorella sp. and Chlorococcum minutum were the most frequently encountered photosynthetic components of biofilms. In all analyzed samples, Trichoderma sp., followed by Cladosporium sp. and Rhizopus stolonifer, were the most frequently identified fungi.Испитиван је диверзитет цијанобактерија, алги и гљива у биофилму с 10 различитих супстрата из стакленика Ботаничке баште „Јевремовац“. Од 37 документованих таксона, идентификовано је 16 цијанобактерија и 10 алги. Преосталих 11 таксона припадају „Петом царству“. Највећа разноврсност микроорганизама, укупно 24 таксона, забележена је у биофилму на кородираној металној површини, док је значајно нижи број таксона регистрован на осталим испитиваним супстратима. Цијанобактерија Porphyrosiphon sp., дијатома Achnanthes sp. и зелене алге Chlorella sp. и Chlorococcum minutum су најчешће фотосинтетичке компоненте биофилма. У свим испитиваним узорцима Trichoderma sp., заједно са Cladosporium sp. и Rhizopus stolonifer су најчешће идентификоване гљиве

Supplementary material for the article: Ilić, B.; Dimkić, I.; Unković, N.; Grbić, M. L.; Vukojević, J.; Vujisić, L.; Tešević, V.; Stanković, S.; Makarov, S.; Lučić, L. Millipedes vs. Pathogens: Defensive Secretions of Some Julids (Diplopoda: Julida) as Potential Antimicrobial Agents. Journal of Applied Entomology 2018, 142 (8), 775–791. https://doi.org/10.1111/jen.12526

Supplementary material for: [https://doi.org/10.1111/jen.12526]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2198

Supplementary data for the article: Ljaljević Grbić, M.; Unković, N.; Dimkić, I.; Janaćković, P.; Gavrilović, M.; Stanojević, O.; Stupar, M.; Vujisić, L.; Jelikić, A.; Stanković, S.; et al. Frankincense and Myrrh Essential Oils and Burn Incense Fume against Micro-Inhabitants of Sacral Ambients. Wisdom of the Ancients? Journal of Ethnopharmacology 2018, 219, 1–14. https://doi.org/10.1016/j.jep.2018.03.003

Supplementary material for: [https://doi.org/10.1016/j.jep.2018.03.003]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2158