Isolation and attempts of biomolecular characterization of fungal strains associated to foxing on a 19th century book

The brownish spots known as foxing, commonly found on old paper artefacts contain sometimes structures, which look like filamentous fungi. Attempts to grow these structures in vitro have been always unsuccessful so far. In order to study the role of these biological elements in paper decay, it is essential to identify them. This study is aimed at the identification of isolates from these brownish areas by culture-independent approaches using molecular biology techniques. The two Internal Transcribed Spacers and the 5.8 S gene (ITS1-5.8S-ITS2) from the nuclear ribosomal DNA were amplified, cloned and sequenced. Following a preliminary treatment with cellulase from Trichoderma reesei, DNA extractions were successfully achieved directly from paper samples. From 22 selected stained spots from a book dating from the 19th century, 8 extracts of genomic DNA were entirely analysed, which yielded 145 sequenced clones. No DNA could be sampled in unstained areas. Multiple alignment of the ITS sequences and comparison with reference sequences published in the NCBI database allowed to identify 14 groups of fungi belonging mainly to the following genera: Aspergillus, Bjerkandera, Chaetomium, Gloeotinia, Penicillium, Polyporus, Saccharicola, Trichoderma and Ulocladium. Some of these fungi are cellulolytic species but are not commonly found as indoor contaminant in storage rooms of graphic documents. The majority of the foxing spots exhibited sequences identified as Penicillium minioluteum. Gloeotinia temulenta occurred also frequently. Few isolates such as Aspergillus japonicus, Aspergillus oryzae, Chaetomium globosum, Penicillium citrinum, Trichoderma citrinoviride, Ulocladium chartarum and Ulocladium cucurbitae were present in only 1 or 2 clones. One of the foxing spots produced only one type of sequence similar to both Cordyceps sinensis and Fusarium lateritium, which have identical sequences in this rDNA region. This molecular approach, simple and rapid, could provide additional data for further discussion on the origin of the phenomenon of biological foxing.