Pedro M. Martin-Sanchez

Detection of human-induced environmental disturbances in a show cave

PurposeWe investigated the effects of human-induced disruption in a subterranean stable environment containing valuable Palaeolithic paintings and engravings (Ardales Cave, Southern Spain) using a double analytical approach.MethodsAn environmental monitoring system was installed in the cave to record temperature, relative humidity, carbon dioxide (CO2) and radon (222Rn) concentrations in air. In the same stations, an aerobiological sampling was conducted to quantify the level of airborne microorganisms.ResultsThe combination of different methods allowed us to detect the extent of human-induced changes, confirming that these can be very hazardous in certain cave areas that should be apparently outside the scope of human disturbances, either by their remoteness to the visitor entrance or by being briefly visited.ConclusionsThe detection of evident anomalies in the environmental parameters and airborne microorganism concentration in the cave area housing the high density of paintings and engravings helps to control human disturbances and supports the direct application of this double approach for cave management purposes.

Use of biocides for the control of fungal outbreaks in subterranean environments: the case of the Lascaux Cave in France

The Lascaux Cave in France suffered an outbreak of the fungus Fusarium solani in 2001. Biocides were applied for three years to control this outbreak. Four months after the initial biocide application, a new outbreak appeared in the form of black stains that progressively invaded the cave. The black stains on the ceiling and passage banks were so evident by 2007 that they became one of the caves major problems. Therefore, biocides were used again in 2008. The present study investigated the fungal communities associated with the black stains and the effectiveness of the biocides applied, by using cloning, denaturing gradient gel electrophoresis, and culture-dependent methods. A novel species, Ochroconis lascauxensis, was the most abundant fungus in samples collected between 2007 and 2008, and the biocides applied were not effective in eliminating this fungus; on the contrary, they appeared to increase the fungal diversity. The fungal communities represented in the samples collected in 2010 were quite different from those collected in 2008 and 2009: the major OTUs corresponded to black yeasts belonging to the Herpotrichiellaceae family. The origin and evolution of these microorganisms are probably linked to the intensive biocide treatments and to the anthropogenic changes introduced by cave management.

Two new species of the genus Ochroconis, O. lascauxensis and O. anomala isolated from black stains in Lascaux Cave, France

In the year 2001, some conspicuous black stains appeared on the walls of Lascaux Cave in France, which progressively disseminated throughout the cave. These black stains were so evident by 2007 that they have become one of the caves major problems. In a mycological study of the black stains, Ochroconis strains were abundant among the isolates and constituted the major group of melanised fungi. Two new species of the genus Ochroconis, O. lascauxensis and O. anomala, were isolated and described. The description is based on the morphology of the fungi and the phylogenetic relationships of two of its gene regions internal transcribed spacer (ITS) and RNA polymerase II subunit B (RPB2). In addition, data on their physiology and cellular fatty acid profiles are reported. The development of these species was likely linked to the presence of unusual carbon and nitrogen organic sources provided by the intensive biocide treatments.

Uncovering the origin of the black stains in Lascaux cave in France

Lascaux Cave in France was discovered in 1940. Since being opened to visitors the cave has suffered three major microbial outbreaks. The current problem is the fast dissemination of black stains which are threatening the Palaeolithic paintings. Previous data pointed to the involvement of new fungal species in the formation of black stains on the rock walls and ceiling. However, it appears that there could be other reasons for the formation of different and extensive black stains coating the surface of the clayey sediments. Our analyses reveal that black stains on clayey sediments are mainly produced by Acremonium nepalense, a manganese oxide-depositing fungus, widely distributed in the cave. Thus, in Lascaux Cave, the black stains have a dual origin: on limestone rocks they are mainly produced by the accumulation of fungal melanins, and on clayey sediments by the biogenic deposition of black manganese oxides.

Real-time PCR detection of Ochroconis lascauxensis involved in the formation of black stains in the Lascaux Cave, France.

A real-time Polymerase Chain Reaction (PCR) assay was developed to detect and quantify Ochroconis lascauxensis in the Lascaux Cave in France. This fungus is the principal causal agent of the black stains threatening the Paleolithic paintings of this UNESCO World Heritage Site. The black stains outbreak could not be stopped in spite of using intensive biocide treatments. A sensitive and time-saving protocol is needed for determining the extent of the colonization. Sets of primers that target the ITS and RPB2 regions were designed and evaluated for specificity against O. lascauxensis. Genomic DNA extracted from five species of Ochroconis and 13 other fungal species frequently isolated from caves were used to test the specificity of each primer set. The specific and sensitive real-time PCR assay using the primers 347F/493R targeting a 147-bp fragment from the RPB2 gene was useful for quantifying the presence of O. lascauxensis in the stains on the walls, sediments and air of the cavity. The results confirmed the association of this fungus with the black stains and its wide dissemination in all cave compartments. The suitability of this method for monitoring fungal outbreaks in cave environments is discussed.

Airborne microorganisms in Lascaux Cave (France)

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Free-living amoebae in sediments from the Lascaux Cave in France

The research was initially supported by the Ministry of Culture and Communication, France, project “Ecologie microbienne de la grotte de Lascaux”. Later, the work was supported by the Spanish Ministry of Science and Innovation, project TCP CSD2007- 00058.

Structure of melanins from the fungi Ochroconis lascauxensis and Ochroconis anomala contaminating rock art in the Lascaux Cave

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.

Roof-Inhabiting Cousins of Rock-Inhabiting Fungi: Novel Melanized Microcolonial Fungal Species from Photocatalytically Reactive Subaerial Surfaces

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.

Diversity and biocide susceptibility of fungal assemblages dwelling in the Art Gallery of Magura Cave, Bulgaria

*pedro-maria.martin-sanchez@bam.de Citation: