Ute Moreth

Identification of indoor rot fungi by taxon-specific priming polymerase chain reaction.

Summary The internal transcribed spacer (ITS) of the nuclear ribosomal DNA (rDNA) of the main fungal species causing wood rot damages in European buildings was amplified by the polymerase chain reaction (PCR). After sequencing the ITS, fungus-specific oligonucleotide primers were designed for taxon-specific priming PCR. These DNA marker molecules were suitable for the differential diagnosis of the Dry rot fungus, Serpula lacrymans, the Wild merulius, S. himantioides, the Oak polypore, Donkioporia expansa, the Brown cellar fungus, Coniophora puteana, the Broad-spored white polypore, Antrodia vaillantii, the Sap polypore, Tyromyces placenta, and the Yellow-red gill polypore, Gloeophyllum sepiarium. Each specific marker identified isolates of its respective target species. Cross reaction with ‘foreign’ fungi was the exception. Species detection from rot samples in buildings was possible, since DNA from contaminating organisms does not response to the marker molecules. The diagnosis was rapid, since preceding fungal pure cultures, special DNA extraction/purification and restriction by endonucleases are not required.

Molecular Identity of Species and Isolates of Internal Pore Fungi Antrodia spp. and Oligoporus placenta

Summary Antrodia vaillantii (DC.: Fr.) Ryv., A. serialis (Fr.) Donk, A. sinuosa (Fr.) P. Karsten, A. xantha (Fr.: Fr.) Ryv. and Oligoporus placenta (Fr.) Gilb.& Ryv. form a group of internal brown-rot fungi (‘Porenschwämme’), which are associated with decay of coniferous woodwork in buildings and timber in ground contact. The fungi have similar occurrence, biology, fruit bodies and mycelia. Their nomenclature has a confusing history and is still not uniform. For a better understanding of the domestic pore fungi and for a reliable species differentiation and identification of isolates, the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) was amplified by polymerase chain reaction and sequenced. Isolates from decayed wood and from culture collections were used. The ITS sequences characteristic of A. vaillantii, A. serialis,A. sinuosa, A. xantha and O. placenta were obtained and deposited in the international databases. The ITS size ranges from 636 to 668 bp. Intraspecific variation was low. A dendrogram was performed for the phylogenetic relationship. Some isolates obtained mislabelled were named correctly according to the ITS sequence. The sequences contribute to our collection of ITS data from internal wood decay fungi made to characterize fungi in culture.

Identification of the dry rot fungus, Serpula lacrymans, and the wild merulius, S. himantioides, by amplified ribosomal DNA restriction analysis (ARDRA)

Summary Isolates of the dry rot fungus, Serpula lacrymans, and of the morphologically similar wild merulius, S. himantioides, were investigated by amplified ribosomal DNA restriction analysis (ARDRA) of the internal transcribed spacer (ITS) region to prove this method as diagnosis tool for the economically important indoor rot fungi. The technique uses the polymerase chain reaction (PCR) to amplify the relatively variable sequences of the ITS region arranged between the highly conserved portions of the 18S and 28S RNA genes of the nuclear ribosomal DNA (rDNA) repeat unit. Subsequent digestion of the amplicon with restriction endonucleases may exhibit differences at species and subspecies level. Using the universal ITS 1/ITS 4 primer combination, the ITS region of all isolates of S. lacrymans and S. himantioides was amplified. The size of the amplified products was about 630bp in both species, as estimated from agarose gel electrophoresis. Digestion of the amplicon with the endonuclease pairs AluI/HhaI and AvaII/MboII, respectively, revealed identical rDNA-ITS fragments for the isolates of both species, indicating their genetic relationship. On the other hand, digestion with BglI/Hinf I and HaeIII/TaqI, respectively, separated the fungi by means of different fragment patterns. Thus, ARDRA-ITS proved to be suited for the identification of both fungi.

Characterization of indoor rot fungi by RAPD analysis

Various isolates of the dry rot fungus, Serpula lacrymans, the wild merulius. S. himantioides, and the cellar fungus. Coniophora puteana, from Asia, Europe and USA were investigated by the polymerase chain reaction (PCR) based technique of randomly amplified polymorphic DNA (RAPD). The banding patterns obtained revealed for S. lacrymans a rather species-specific reaction which may be used to distinguish the dry rot fungus from other indoor rot species. On the other hand. the isolates of the wild merulius and the cellar fungus showed more polymorphisms possibly suitable for identification of individual isolates.

Wood Decay by the White-Rotting Basidiomycete Physisporinus vitreus from a Cooling Tower

Timbers in cooling towers are mainly attacked by soft rot causing fungi which belong to Ascomycetes and Deuteromycetes. The basidiomycete Physisporinus vitreus degraded water-saturated limber as fibrous white-pocket rot in a cooling tower in which water treatment had been changed from chlorine to ozone. In the laboratory, the fungus revealed a remarkable wood decay pattern. In crosswise piled. water-saturated pine specimens it attacked only those parts not surrounded by air. The decay occurred as small longish delignified white pockets, preferentially in the earlywood. Transmission electron microscopy of unstained sections showed some electron density in the hyphal extracellular layer and in the wood cell wall beneath a hypha. Contrasted with KMnO 4 staining these regions became more pronounced which may indicate presence of lignin depredation products. UV-microspectrophotometry of these areas exhibited an increased absorbance. Many decay pockets were black due to manganese (Mn) deposits. Mn determination by inductively coupled plasma emission (ICP) revealed up to 518 ppm Mn. TEM/EDXA showed Mn deposits in the hyphal extracellular layer, on the cell wall surface and in the inner S 2 layer beneath a hypha. The Mn may he related to the lignin attacking peroxidases.

Biological Characterization of Poria Indoor Brown-Rot Fungi

Pure cultures of the Paria indoor brown-not fungi, Antrodia vaillantii, A.sinuosa, A.serialis, A.xantha and Tyromyces placenta, were studied regarding growth rate, response to temperature, copper tolerance, wood decay and mycelial interactions. Radial growth extension reached from 4-9 mm/d. All fungi were rather resistant to high temperatures. Optimum temperature was between 25 and 31°C. All withstood 1 hour at 60°C and some even 3h at 65°C. A.vaillantii showed copper tolerance up to 0.05 M Cu. Wood weight loss after 20 weeks was higher by T.placenta (35%) and A.sinuosa (33%) than by A.xantha (21%), A.serialis (16%) and A.vaillantii (14%). Dual cultures showed various inter- and intraspecific interactions and detected identity of differently coded cultures. The former Poria vaporaria Normstamm II for testing wood preservatives and the recent Poria placenta EN113 strain, FPRL280, were assumed either to be identical or at least to originate from the same individual.

Molecular identity of species and isolates of the Coniophora cellar fungi

Summary Within the genus Coniophora, C. puteana and the less common species C. marmorata, C. arida and C. olivacea form a group of domestic brown-rot fungi (“cellar fungi”), which cause considerable decay in the woodwork of buildings. The fungi are difficult to distinguish by their fruit bodies. Traditional methods fail to identify species in a pure culture. Also with regard to decay of wood in use, the basidiomes rarely develop, and the surface mycelium on wood is sparse except for the fine dark strands. Thus, molecular techniques were applied to obtain a reliable method for differentiation and detection and to aquire a greater knowledge of the domestic Coniophora species. Isolates obtained from decayed wood and from culture collections and identified as domestic Coniophora species were used. The internal transcribed spacer (ITS) of the ribosomal DNA was amplified by polymerase chain reaction, sequenced and restricted by the endonuclease TaqI. The ITS sequences characteristic of C. puteana, C. arida, C. marmorata and C. olivacea were obtained and entered into the international databases. The sequence size ranges from 525 to 729 bp. The data supplement our collection of ITS sequences from domestic wood decay fungi built for the characterization of mycelium in culture. Most isolates belong to C. puteana and some to C. arida, C. marmorata and C. olivacea. Two other isolates belong to a further Coniophora species close to C. olivacea. Obviously, at least five Coniophora species occur within European buildings. Many of the isolates were not correctly identified. Consequently, the traditional characterization methods used, viz., growth rate, temperature influence and wood weight loss, were less suitable for the differentiation of Coniophora species as formerly anticipated.

Investigations on ribosomal DNA of indoor wood decay fungi for their characterization and identification

cause wood decay in buildings. Withregard to remedial treatment of the damage caused bythese fungi and for scientific purposes, species identityshould be known. An identification key on the basis offruit bodies is in the Internet (Huckfeldt 2002), and a keywas developed for species that form mycelial cords(Huckfeldt and Schmidt 2004). However, often only veg-etative mycelium is found in buildings. Furthermore,experience has shown that some cultures held in straincollections need re-identification (Schmidt et al. 2002;Schmidt and Moreth 2003a). Keys based on morpholog-ical and physiological characters of the vegetative myce-lium cannot discriminate closely related species. Thus,molecular methods were used for wood decay fungi (Pal-freyman 1998; Schmidt 2000; Jasalavich et al. 2000;Adair et al. 2002; Diehl et al. 2004). SDS-PAGE of pro-teins discriminated

Improved waterproofing of UF plywood adhesives by melamine salts as glue mix hardeners: system performance optimization.

Species of the Poria group causing brown rot of wood in buildings and with similar morphological appearance and biology, namely Antrodia vaillantii, A. seriadis, A. sinuosa, A. xantha, and Tyromyces placenta, were investigated by SDS polyacrylamide gel electrophoresis. Different cultures of A. vaillantii showed a species-specific protein profile separating the fungus from A. serialis, A. sinuosa and A. xantha and from T. placenta. The method also differentiated these pore fungi from the dry rot fungus Serpula lacrymans and detected misidentifications

Ribosomal DNA intergenic spacer of indoor wood-decay fungi

Abstract Indoor wood-decay fungi are economically very important. Approximately half of the total damage caused by indoor fungi in Germany is caused by species of the Coniophoraceae. The sequence of the intergenic spacer (IGS) region of the ribosomal DNA was elucidated with the following fungi of this family: Serpula lacrymans (true dry rot fungus), S. himantioides (wild merulius), Meruliporia incrassata (North American dry rot fungus), Leuco-gyrophana pinastri (mine dry rot fungus), Coniophora puteana (brown cellar fungus), and C. marmorata (marmoreus cellar fungus). The IGS length ranges between 2584 and 3785 bp and consists of the short IGS 1 (253–440 bp), the 5S rDNA (118 bp) and the long IGS 2 (2193–3310 bp). IGS 1 is phylogenetically less informative for the investigated Coniophoraceae species. 5S rDNA is transcribed in the reverse direction. IGS 2 contains extended repeat blocks of copies of different length. Intraspecific length polymorphism as a result of different copy number occurs in M. incrassata and L. pinastri. In combination with previous results, the full-length sequence of the rDNA repeat unit is available for important indoor wood-decay fungi. The various rDNA regions can now be used for future identification of unknown sequences by BLAST and also for phylogenetic studies.