Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis

Abstract

Besides lichens and bacteria, fungi play a crucial role in the biodeterioration of historical glass. In the present paper, the fungal diversity on the surface of two historical church window glasses was investigated by 18S rDNA-based denaturing gradient gel electrophoresis (DGGE) analysis. 566-bp 18S rDNA-specific clone libraries were constructed with primer set NS1/NS2+10. Positive clones were reamplified with primer sets EF4/518rGC (426-bp fragments) and NS26/518rGC (316-bp fragments), amplicons were screened by DGGE and clustered according to their position in DGGE. Results indicated that fungal 18S rDNA clone libraries should be screened with at least two different primer sets to obtain the maximum number of different clones. For phylogenetic sequence analyses, clone inserts were sequenced and compared with 18S rDNA sequences listed in the EMBL database. Similarity values ranged from 93.7% to 99.81% to known fungi. Analyses revealed complex fungal communities consisting of members and relatives of the genera Aspergillus, Aureobasidium, Coniosporum, Capnobotryella, Engyodontium, Geomyces, Kirschsteiniothelia, Leptosphaeria, Rhodotorula, Stanjemonium, Ustilago, and Verticillium. The genera Geomyces and Aureobasidium were present on both glass surfaces. Some genera had not been detected on historical glass so far.

Introduction

The biocorrosion of antique and optical glass is a well-known phenomenon. It is presumed that biodeterioration is the result of metabolic activities of complex microbial communities composed of lichens (Mellor, 1924), fungi Nagamuttu, 1967, Kerner-Gang and Schneider, 1969, Tennent, 1981, Kaiser et al., 1996 and bacteria (Rölleke et al., 1999). Not the inorganic material of the glass itself facilitates microbial growth, but the organic residues of various origin which are always present on historical glass, such as dust deposits, dead fungal and bacterial material, metabolites of autotrophic bacteria and animal faeces. The biodeteriorative role of microorganisms includes both chemical and mechanical destruction of glass. A leaching environment created by the adsorption of water by actinomycetal and fungal mycelium enhances chemical destruction. The excretion of aggressive metabolic products such as organic or inorganic acids leads to changes in pH, to oxidation, reduction, leaching and chelation of special glass components. In consequence, microbial growth on glass surfaces enhances further mechanical biodeterioration such as etching, pit corrosion, crack formation and patina formation Krumbein et al., 1991, Drewello and Weissmann, 1997.

Investigations on fungal colonization of historical glass are based on cultivation studies so far. In these studies, mainly species of the genera Alternaria, Aspergillus, Aureobasidium, Cladosporium, Chaetomium, Engyodontium, Eurotium, Fusarium, Monodictys, Mucor, Penicillium, Phoma, Rhizopus, Scopulariopsis Stemphilium Trichoderma, Ulocladium, and Verticillium were cultivated from glass surfaces Theden and Kerner-Gang, 1964, Krumbein et al., 1991, Drewello and Weissmann, 1997. For appropriate measurements against microbial growth on objects of art such as historical glass and wall paintings, it is important to obtain an overview of the present microbial populations. In general, these investigations cover only those microorganisms that can be cultivated Giovannoni et al., 1990, Ward et al., 1990. As microorganisms on objects of art are mostly members of a complex microbial consortium and depend on special nutrients, only a minority thereof can be cultivated under laboratory conditions. As cultivation-independent methods enable the detection of slowly growing, fastidious or uncultivable microorganisms, it is likely that molecular methods give a more complete view of the present microbial community than traditional cultivation techniques. When working with samples taken from art objects, only small quantities of sample material are available which are sometimes not efficient enough for cultivation experiments. On the contrary, molecular-based techniques require little sample material in the range of 1–2 mg (Schabereiter-Gurtner et al., 2001).

The application of 16S rDNA-based community fingerprinting by denaturing gradient gel electrophoresis (DGGE) helped to understand the biodiversity of bacterial communities in natural samples and revealed much more complex communities than could be shown by cultivation (Muyzer et al., 1993). The DGGE approach allows the separation of PCR amplicons of same length according to their sequence. During gel electrophoresis, short PCR amplicons migrate towards increasing denaturing concentrations, leading to a partial melting of the DNA helix and to a decrease and subsequent haltering of electrophoretic migration. As a consequence, a banding pattern is produced, in which each band represents a microbial taxon. The sequence-specific separation of ribosomal PCR amplicons facilitates therefore the profiling of monomicrobial as well as polymicrobial communities. By either excising individual DGGE bands from the gel and extracting and reamplifying the DNA, or by construction of clone libraries and screening clones by DGGE, it is possible to get phylogenetic sequence information of individual microbial members of the microbial community.

While 16S rDNA DGGE fingerprinting is hitherto broadly used for monitoring bacterial growth and analyzing bacterial communities, the analysis of fungal communities by 18S rDNA DGGE fingerprinting is so far not well established. The aim of the present study was to develop a protocol for 18S rDNA-based DGGE fingerprinting and apply it to the investigation and comparison of the fungal diversity on two historical church window glasses. For this purpose, 18S rDNA clone libraries were constructed, clones were reamplified with two different primer sets and screened by DGGE. Individual fungi were phylogenetically classified by comparison of sequences with sequences listed in the EMBL database.