RESUMEN
The Capuchin Catacombs of Palermo, Italy, contain over 1800 mummies dating from the 16th to 20th centuries AD. Their environment is not conducive to the conservation of the remains due to, among other factors, water infiltration, which is producing salt efflorescences on the walls. A multiphasic approach was applied to investigate the halophilic microbiota present in the Catacombs. Enrichment cultures were conducted on media containing different NaCl concentrations, ranging from 3 to 20 %. For screening of the strains, the following two PCR-based methods were used and compared: fluorescence internal transcribed spacer PCR (f-ITS) and random amplification of polymorphic DNA (RAPD) analyses. Results derived from RAPD profiles were shown to be slightly more discriminative than those derived from f-ITS. In addition, the proteolytic and cellulolytic abilities were screened through the use of plate assays, gelatin agar and Ostazin Brilliant Red H-3B (OBR-HEC), respectively. Many of the strains isolated from the wall samples displayed proteolytic activities, such as all strains belonging to the genera Bacillus, Virgibacillus and Arthrobacter, as well as some strains related to the genera Oceanobacillus, Halobacillus and Idiomarina. In addition, many of the strains isolated from materials employed to stuff the mummies showed cellulolytic activities, such as those related to species of the genera Chromohalobacter and Nesterenkonia, as well as those identified as Staphylococcus equorum and Halomonas sp. Furthermore, many of the strains were pigmented ranging from yellow to a strong pink color, being directly related to the discoloration displayed by the materials.
Asunto(s)
Bacterias/aislamiento & purificación , Cuevas/microbiología , Microbiota , Momias/microbiología , Tolerancia a la Sal , Bacterias/genética , Bacterias/metabolismo , Italia , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
In this study, we investigated and compared the microbial communities adhering to the obverse and the reverse sides of an oil painting on canvas exhibiting signs of biodeterioration. Samples showing no visible damage were investigated as controls. Air samples were also analysed, in order to investigate the presence of airborne microorganisms suspended in the indoor atmosphere. The diversity of the cultivable microorganisms adhering to the surface was analysed by molecular techniques, such as RAPD analysis and gene sequencing. DGGE fingerprints derived from DNA directly extracted from canvas material in combination with clone libraries and sequencing were used to evaluate the non-cultivable fraction of the microbial communities associated with the material. By using culture-dependent methods, most of the bacterial strains were found to be common airborne, spore-forming microorganisms and belonged to the phyla Actinobacteria and Firmicutes, whereas culture-independent techniques identified sequenced clones affiliated with members of the phyla Actinobacteria and Proteobacteria. The diversity of fungi was shown to be much lower than that observed for bacteria, and only species of Penicillium spp. could be detected by cultivation techniques. The selected strategy revealed a higher microbial diversity on the obverse than on the reverse side of the painting and the near absence of actively growing microorganisms on areas showing no visible damage. Furthermore, enzymatic activity tests revealed that the most widespread activities involved in biodeterioration were esterase and esterase lipase among the isolated bacterial strains, and esterase and N-acetyl-ß-glucosaminidase among fungi strains.
RESUMEN
The nuclear rRNA gene of Ophiostoma piliferum was analyzed to understand its phylogenetic relationships to other sapstain fungi. Phylograms based on nucleotide sequences of the rRNA gene showed that the relationships between O. piliferum and other Ophiostoma species varied depending on the regions of the rRNA gene analyzed. Intraspecies variation in O. piliferum was found in the internal transcribed spacer regions, and the variation was related to the geographic origin of O. piliferum strains. A useful molecular marker for differentiating O. piliferum from other sapstain Ophiostoma species was generated by the HaeIII restriction fragment length polymorphism of the 26S rRNA gene.
Asunto(s)
Ascomicetos/clasificación , Ascomicetos/genética , Genes de ARNr , Filogenia , ADN de Hongos/genética , ADN Ribosómico/genética , ADN Espaciador Ribosómico/genética , Genes Fúngicos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico/genéticaRESUMEN
The diversity of fungal communities on three different historical monuments in the city of Vienna (Austria) was analyzed and compared to the fungal diversity of microfungi on rock in the original quarry located in a rural area (Zogelsdorf, Austria). The fungal strains isolated were characterized by morphology and the complete rock fungal community was identified based on molecular data, that is, by sequencing parts of the small ribosomal subunit (18S) and internal transcribed spacer region 1 (ITS1). The genera Coniothyrium, Epicoccum and Phoma were found to be dominant on monument and rock surfaces. Additionally, black yeasts such as Exophiala species and microcolonial fungi like Sarcinomyces and Coniosporium which hitherto were regarded as typical rock inhabitants in semi-arid environments are frequently found on all rock surfaces in Vienna. The biodiversity of the fungi in the urban environment was much higher than on the same rock type in a rural environment, this difference can be attributed to the elevated organic pollution in the city.
Asunto(s)
Microbiología Ambiental , Hongos/clasificación , Hongos/genética , Austria , Secuencia de Bases , Cladosporium/clasificación , Cladosporium/genética , Cladosporium/aislamiento & purificación , Cartilla de ADN/genética , ADN de Hongos/genética , ADN Intergénico/genética , ADN Ribosómico/genética , Ecosistema , Hongos/aislamiento & purificación , Filogenia , Reacción en Cadena de la Polimerasa , Población Rural , Microbiología del Suelo , UrbanizaciónRESUMEN
We used digoxigenin-labeled probes for in situ hybridization of hyphomycetes to replace the commonly used fluorescent proof of probe binding by a colorimetric reaction. The resulting blue-purple, intracellular precipitate could be easily detected by light microscopy, and thus presented a promising method to overcome autofluorescence of fungal material and substratum. Optimal cell fixation and permeabilization procedures, as well as hybridization conditions were developed on the example of two different hyphomycetes: Phialophora sp. and Cartapip, a colorless mutant of Ophiostoma piliferum (Agra Sol).
Asunto(s)
Colorimetría/métodos , ADN de Hongos/análisis , Hibridación in Situ/métodos , Hongos Mitospóricos/aislamiento & purificación , Sondas de Oligonucleótidos , Precipitación Química , Digoxigenina , Fluorescencia , Hongos Mitospóricos/genética , Phialophora/genética , Phialophora/aislamiento & purificación , Fijación del TejidoRESUMEN
A protocol for application of Polymerase Chain Reaction (PCR) in situ hybridization for the detection of hyphomycetes is presented. The experiments are exemplary carried out with strains of the genera Penicillium and Cladosporium. The small ribosomal subunit is amplified in situ by PCR using fungal specific primers. The amplicon is used as target region for a fluorescein-marked probe. The permeability of the fungal cell wall for the primers and the probe can be successfully achieved by enzymatic treatment with beta-glucanase. The protocol can be used as a basis for further development of in situ hybridization with taxon specific probes.
Asunto(s)
Hibridación in Situ/métodos , Hongos Mitospóricos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Black meristematic fungi together with lichens and cyanobacteria dominate the micro-flora of rock surfaces in arid and semi-arid environments of hot and cold deserts. This study shows that rock inhabiting meristematic fungi are extremely tolerant against high temperatures, desiccation and osmotic stress. Their temperature tolerance increases with increasing dehydration of the fungal thallus. Air dried mycelia of black yeasts stand temperatures up to 120 degrees C for at least 0.5 hours. As response to high temperatures multilayered cell walls are developed and trehalose is accumulated whereas the intracellular glycerol regulates the osmotic potential under NaCl stress. Strains from rock in moderate climate (North Germany) show the same tolerance than those isolated from the Mediterranean area. Hortaea werneckii--hitherto only described as agent of human Tinea nigra--is shown to be the most tolerant rock inhabiting species tested. Meristematic fungi cannot be pre-adapted to higher growth temperatures by increased incubation temperatures. Considering the results of this study the justification of the term 'stress' is discussed with regard to rock inhabiting fungi and their natural environment. Consequences for conservation treatments of monuments decayed by meristematic fungi are discussed on the basis of the ecophysiological properties of the fungi.
Asunto(s)
Microbiología Ambiental , Hongos Mitospóricos/fisiología , Desecación , Ecología , Fenómenos Geológicos , Geología , Glicerol/análisis , Calor , Hongos Mitospóricos/ultraestructura , Presión Osmótica , Cloruro de Sodio , Trehalosa/análisisRESUMEN
Coniosporium perforans and C. apollinis, originating from marble in the Mediterranean basin, are described as new species of rock inhabiting microcolonial fungi. The morphologically similar species Monodictys castaneae (Wallr.) S. Hughes, Phaeosclera dematioides Sigler et al., and a Coniosporium-like strain are compared using 18S rDNA phylogeny and Restriction Length Fragment Polymorphism analysis of Internal Transcribed Spacer regions. Sarcinomyces crustaceus Lindner is additionally compared on the basis of 18S rDNA sequencing data. Phylogenetic analysis suggests that Phaeosclera dematioides is related to the ascomycetous order Dothideales and Monodictys castaneae to the Pleosporales, whereas the three Coniosporium species studied are a sister group to the Herpotrichiellaceae (Chaetothyriales). A similar affinity was suggested previously for the recently described meristematic rock-fungus Sarcinomyces petricola Wollenzien & de Hoog. Sarcinomyces crustaceus appears unrelated to this group, and hence the present new taxa cannot be described in this genus.