Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects

A protocol for efficient extraction of fungal DNA from micromycetes colonising painted art objects was developed. Polymerase chain reaction (PCR) inhibitors were successfully removed by a combined application of a Chelex-100 adsorption resin and a Geneclean Kit for Ancient DNA. Universal fungal primers for PCR amplification of 28S rDNA (U1 and U2) were tested for their applicability in denaturing gradient gel electrophoresis (DGGE) analysis of fungal communities. Artificially produced mortar samples inoculated with fungal pure cultures isolated from mural paintings were used as model objects for DNA extractions and DGGE analysis. Good resolution in DGGE was achieved using 260-bp rDNA fragments amplified with U1/DGGE and U2 primers directly from model communities.     

Sensitivity and specificity of phenoloxidase reactions of 1059 strains and species of Micromycetes cultivated on malt/agar medium

The phenoloxidase (POx) activities of 1059 strains and species of micromycetes were determined on malt/agar medium. Overall, 600 (57%) of the isolates produced one or more POx. The sensitivity and specificity of the POx activities towards various substrates were used to group the isolates. Some 187 strains (31% of those producing POx) produced well-defined enzymes, 236 (39%) produced incompletely identified enzymes and 177 (30%) produced other, unidentified POx.     

Production of patulin by Micromycetes

In the context of the screening for antifungal and antibacterial molecules among the Micromycetes, we investigated the ability of 850 strains grown in liquid medium to produce patulin. The compound was produced by 58 fungi, most of which had not been mentioned in the literature, especially Mucorales and Fungi Imperfecti. Biological tests showed that dermatophytes are the most sensitive to this molecule. It also appears that the origin of the producing strains influences their toxin producing activity.     

Metabolism of vanillic acid by Micromycetes

The ability of 953 strains of Micromycetes to grow with vanillic acid (0.5 g/l) was investigated. Toxicity assays were performed on malt extract/agar medium, while consumption was estimated by growing fungi on solid synthetic medium with vanillic acid as sole carbon source. More than half of the tested strains grew in both conditions. After cultivation on solid media, 296 strains were selected and cultivated in liquid synthetic medium. These experiments allowed division of the Micromycetes into different groups according to their consumption of the phenolic compound and the appearance of new metabolites. Results were related to the taxonomic position of the strains.     

Biodeterioration of some herbal raw materials by storage fungi and aflatoxin and assessment of Cymbopogon flexuosus essential oil and its components as antifungal

Deterioration of raw materials of six medicinal plants viz. Terminalia arjuna, Acorus calamus, Rauvolfia serpentina, Holarrhena antidysenterica, Withania somnifera and Boerhaavia diffusa was examined. Some of the contaminated raw materials were found to be deteriorated by toxigenic strains of Aspergillus flavus and contain aflatoxin B₁ (41.0–95.4 μg kg⁻¹) which is above the permissible limit. Essential oil of Cymbopogon flexuosus and its components was found efficient in checking fungal growth and aflatoxin production. C. flexuosus essential oil absolutely inhibited the growth of A. flavus and aflatoxin B₁ production at 1.3 μl ml⁻¹ and 1.0 μl ml⁻¹ respectively. The individual oil components were more efficacious than the Cymbopogon oil as such which emphasizes masking of their efficacy when combined together. Eugenol exhibited potent antifungal and aflatoxin inhibitory activity at 0.3 μl ml⁻¹ and 0.1 μl ml⁻¹ respectively. Eugenol was found superior over some prevalent synthetic antimicrobials and exhibited broad fungitoxic spectrum against some biodeteriorating moulds. Prospects of exploitation of the oil and its components as acceptable plant based antimicrobials in qualitative as well as quantitative control of biodeterioration of herbal raw materials have been discussed.     

In vitro inhibition of Helicobacter pylori by micromycetes

The anti-Helicobacter pylori effect of 22 micromycetes were studied against one standard strain and 11 clinical isolates of H. pylori. Penicillium ochlochloron and Penicillium funiculosum have been proven the most active fungi against this microorganism. Further bio-guided chemical analysis of P. funiculosum afforded an active component identified as (-) 2,3,4-trihydroxybutanamide.     

Distribution of microorganisms on ancient wall paintings as related to associated faunal elements

Knowledge of the bioreceptivity of wall paintings to microbially induced decay processes has increased considerably in the last few years. Indoor wall paintings are widely recognised as a favourable environment for microbial biofilms in general and for microscopic fungi in particular. This environment also provides a suitable substrate for insects, spiders and other arthropodas. A combination of mycological and entomological studies of indoor wall paintings demonstrates an important ecological correlation between fungi and arthropods in the developmental processes of this complex microbial community. The co-existence of microscopic fungi and arthropods on the surface of wall paintings suggests an ecological interaction between them. Faunal impacts on the growth of biofilms due to grazing and propagule dispersal cannot be overlooked. Along with the above-mentioned perturbations, arthropods can directly damage the wall paintings by bioabrasion during grazing on the biofilm, as well as by the production of dwelling cavities. In this publication, we present the ecological connections between the major microscopic inhabitants of these important objects of cultural heritage. The additional role of arthropods as a remarkable source of organic substrate (e.g., chitin, spidersilk and faecal pellets) available for many micromycetes is discussed.