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Subaerial biofilms (SAB) are an important factor in weathering, biofouling, and biodeterioration of bare rocks, building materials, and solar panel surfaces. The realm of SAB is continually widened by modern materials, and the settlers on these exposed solid surfaces always include melanized, stress-tolerant microcolonial ascomycetes. After their first discovery on desert rock surfaces, these melanized chaetothyrialean and dothidealean ascomycetes have been found on Mediterranean monuments after biocidal treatments, Antarctic rocks and solar panels. New man-made modifications of surfaces (e.g., treatment with biocides or photocatalytically active layers) accommodate the exceptional stress-tolerance of microcolonial fungi and thus further select for this well-protected ecological group. Melanized fungal strains were isolated from a microbial community that developed on highly photocatalytic roof tiles after a long-term environmental exposure in a maritime-influenced region in northwestern Germany. Four of the isolated strains are described here as a novel species, Constantinomyces oldenburgensis, based on multilocus ITS, LSU, RPB2 gene phylogeny. Their closest relative is a still-unnamed rock-inhabiting strain TRN431, here described as C. patonensis. Both species cluster in Capnodiales, among typical melanized microcolonial rock fungi from different stress habitats, including Antarctica. These novel strains flourish in hostile conditions of highly oxidizing material surfaces, and shall be used in reference procedures in material testing.
Two novel species of the fungal genus Ochroconis, O. lascauxensis and O. anomala have been isolated from the walls of the Lascaux Cave, France. The interest in these fungi and their melanins lies in the formation of black stains on the walls and rock art which threatens the integrity of the paintings. Here we report solid-state cross polarization magic-angle spinning 13C and 15N nuclear magnetic resonance (NMR) spectroscopy and surface-enhanced Raman spectroscopy (SERS) of the melanins extracted from the mycelia of O. lascauxensis and O. anomala in order to known their chemical structure. The melanins from these two species were compared with those from other fungi. The melanins from the Ochroconis species have similar SERS and 13C and 15N NMR spectra. Their chemical structures as suggested by the data are not related to 3,4-dihydroxyphenylalanine, 5,6-dihydroxyindole or 1,8-dihydroxynaphthalene precursors and likely the building blocks from the melanins have to be based on other phenols that react with the N-terminal amino acid of proteins. The analytical pyrolysis of the acid hydrolysed melanin from O. lascauxensis supports this assumption.
Contribution of culture-independent methods to the cave aerobiology; the case of Lascaux Cave
(2014)
A novel qPCR protocol to the specific detection and quantification of Hormoconis resinae in fuel systems
P.M. Martin-Sanchez1 , J. Toepel1, H.J. Kunte1, A.A. Gorbushina1, 2
1BAM Federal Institute for Materials Research and Testing, Department 4 Materials & Environment, Berlin, Germany
2 Free University of Berlin, Department of Earth Sciences & Biology, Chemistry & Pharmacy, Berlin, Germany
Introduction. Microbial contamination of fuels is a phenomenon widely reported in the literature causing dramatic problems in fuel systems such as blockage of pipelines and filters, and corrosion of their metal components. A variety of microorganisms, fungi, yeasts and bacteria, can contaminate the distillate fuels. Between them, the filamentous fungus Hormoconis resinae, previously described as Cladosporium resinae and also known as "kerosene fungus" or "diesel bug", has traditionally been considered the main responsible of such troubles due to its great capacity to produce biomass and biofilms.
Aim. The overarching goal of this study was to develop a novel real-time quantitative PCR (qPCR) method to detect and quantify the fungus H. resinae in samples from fuel systems.
Materials and methods. Four specific primer sets targeting on two molecular markers, RNA polymerase II second largest subunit (RPB2) and internal transcribed spacers of rDNA (ITS), were designed using Primer Blast. The specificity of primer sets was evaluated by both conventional PCR and qPCR, analyzing the DNA extracts from H. resinae strains and other fungi and yeasts previously described in fuels as well as four taxonomically closest related species. The efficiency and sensitivity of these protocols were assessed by constructing the corresponding standard curves for each primer sets.
Results.
Two primer sets, Hr556F/Hr814R (RPB2) and Hr101F/Hr408R (ITS), were selected according to their remarkable specificity. Their standard curves showed a good correlation coefficient and efficiency. Additionally, the selected primer pairs showed a high sensitivity detecting a tiny amount of H. resinae DNA, 10 pg for RPB2 primers and 0.1 pg for ITS primers.
Conclusions.
The established qPCR protocol is a reliable and useful tool to the specific detection and quantification of H. resinae. Its subsequent implementation to analyze samples of fuels or biofilms covering fuel systems will allow the early detection of fungal outbreaks in order to apply the appropriated control procedures. Moreover, these analyses will determine the current incidence of this species in the cases of biological fuel contamination.
Keywords. Hormoconis resinae, real-time qPCR, diesel bug, kerosene fungus