Isolation and characterization of 15 polymorphic microsatellite markers for the devastating dry rot fungus,Serpula lacrymans

Fifteen polymorphic microsatellite markers were developed from microsatellite‐enriched DNA libraries of the devastating dry rot fungus, Serpula lacrymans. The loci exhibited two to four alleles per locus and the expected heterozygosity ranged from 0.13 to 0.47. The codominant markers, described here for this fungus, will permit further studies in population genetics and phylogeography of this economically highly important species.     

Evolutionary origin,worldwide dispersal,and population genetics of the dry rot fungus Serpula lacrymans

Recent research on the evolution, phylogeography and population genetics of the dry rot fungus Serpula lacrymans is reviewed. The fungus causes severe damage to construction wood in temperate regions worldwide. Multi-locus genetic analyses have shown that S. lacrymans includes two cryptic species currently referred to as var. shastensis and var. lacrymans. Both lineages occur naturally in high altitude regions, but var. lacrymans has also spread from its natural range in Asia, and established itself in indoor environments in temperate regions worldwide. Japan was apparently colonized independently from Europe, North and South America by var. lacrymans. The population found in Australia and New Zealand seems to represent a mixture of the Japanese and the Euro-American lineages. Little genetic variation exists in the founder populations of var. lacrymans worldwide. Due to the introduction of a limited number of vic (vegetative incompatibility) alleles into the founder populations, genetically different individuals often cannot recognize self from non-self through the vegetative incompatibility response. Moreover, only a low number of MAT (mating) alleles seem to be present in Europe. Genetic analyses of the European and Japanese indoor populations have shown that S. lacrymans mainly spreads sexually via basidiospores. Surprisingly, an excess of heterozygotes has been observed in the founder populations. This could be due to heterozygote advantage caused by a limited number of MAT alleles. Recent analyses of the S. lacrymans genome provide new opportunities for further research on the dry rot fungus.     

Serpula lacrymans reacts with a general,unspecialized chemical response during interaction with mycoparasitic Trichoderma spp. and bacteria

Serpula lacrymans causes structural damage in timber via cellulose and hemicellulose decomposition. Serpula lacrymans shares its habitat with bacteria and fungi, interactions that often result in stress and changed behaviour of the partners. A typical response to environmental stress is the production of bioactive metabolites and pigments. The diversity and function of these metabolites in inter- and intra-kingdom interactions, is largely elusive. Using dual culture approaches and secondary metabolite fingerprinting with high performance thin layer chromatography (HPTLC) it was demonstrated that stress induces an unspecific vs. Similar metabolite patterns were identified when S. lacrymans was co-cultured with different bacteria and mycoparasitic fungi (Trichoderma spp.). By analysing the metabolites produced along the gradient from the contact zone to more remote parts of the mycelium, we show a systemic reaction of S. lacrymans and that direct contact with other microbes is not a necessity to induce secondary metabolites.     

Modelling the effect of temperature, water activity and pH on the growth of Serpula lacrymans

Aims: To predict the risk factors for building infestation by Serpula lacrymans, which is one of the most destructive fungi causing timber decay in buildings. Methods and Results: The growth rate was assessed on malt extract agar media at temperatures between 1·5 and 45°C, at water activity (a_w) over the range of 0·800–0·993 and at pH ranges from 1·5 to 11·0. The radial growth rate (μ) and the lag phase (λ) were estimated from the radial growth kinetics via the plots radius vs time. These parameters were then modelled as a function of the environmental factors tested. Models derived from the cardinal model (CM) were used to fit the experimental data and allowed an estimation of the optimal and limit values for fungal growth. Optimal growth rate occurred at 20°C, at high a_w level (0·993) and at a pH range between 4·0 and 6·0. The strain effect on the temperature parameters was further evaluated using 14 strains of S. lacrymans. The robustness of the temperature model was validated on data sets measured in two different wood‐based media (Quercus robur L. and Picea abies). Conclusions: The two‐step procedure of exponential model with latency followed by the CM with inflection gives reliable predictions for the growth conditions of a filamentous fungus in our study. The procedure was validated for the study of abiotic factors on the growth rate of S. lacrymans. Significance and Impact of the Study: This work describes the usefulness of evaluating the effect of physico‐chemical factors on fungal growth in predictive building mycology. Consequently, the developed mathematical models for predicting fungal growth on a macroscopic scale can be used as a tool for risk assessment of timber decay in buildings.     

Two invasive populations of the dry rot fungus Serpula lacrymans show divergent population genetic structures

The dry rot fungus Serpula lacrymans is a devastating basidiomycete occurring in wooden constructions in temperate regions worldwide. In this study, we compare the genetic structures of two invasive populations from Europe and Japan. Microsatellite data from 14 loci and DNA sequences from four loci demonstrated that the two populations were highly differentiated. Significant isolation by distance effect was observed in Europe and Japan. Higher genetic variation was observed within the Japanese population than within the European population, corresponding with the observed higher richness of vegetative compatibility types in Japan, indicating that there has been a higher level of gene flow from the Asian source populations to Japan than to Europe. The European population is genetically more homogenous with only six detected vegetative compatibility types. Various tests indicate that both the European and the Japanese populations have gone through population bottlenecks prior to population expansion. No identical multi‐locus genotypes were observed within Japan and very few within Europe, indicating limited clonal dispersal. Deviations from Hardy Weinberg expectations were observed both in Europe and Japan and heterozygote excess were observed at several loci, especially in Europe. Possible explanations for this pattern are discussed.     

Wood decay fungi and their bacterial interaction partners in the built environment – A systematic review on fungal bacteria interactions in dead wood and timber

Microbial biodeterioration of timber and woody material in buildings can cause costly restoration procedures. Here, we focus on Serpula lacrymans (commonly known as dry-rot) the fungus causing the most severe damages to buildings in Europe. Although its morphology, lifestyle, and dispersal have been intensively studied, research on microorganisms sharing the same habitat and interacting with the dry-rot fungus is not as comprehensive. Bacteria have long been known to inhabit dead wood, and several studies have shown their association to fungi. However, their identity, ecology, and putative interactions with co-existing fungi in dead wood remains largely underexplored. The interactions of bacterial and fungi have considerable impact on all partners involved covering the full spectrum between antagonistic and beneficial. Fungi are highly capable of manipulating the microbial community in their surroundings (e.g. via pH manipulation) and bacteria, in turn, can influence fungi by affecting the outcomes of (antagonistic) interactions or preventing fungal feedback inhibition via consumption of breakdown products. Associated bacteria on the other side could play an essential role for the fungus as bacteria can exert significant influence on fungal physiology and behaviour. This minireview summarizes the current knowledge on bacterial-fungal interactions in dead wood with a special focus on dry-rot and proposes possible bacterial-fungal interaction (BFI) mechanisms based on examples from soil or decomposing wood from forests.     

Filamentous fungal characterizations by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight intact cell mass spectrometry (MALDI‐TOF ICMS) is coming of age for the identification and characterization of fungi. The procedure has been used extensively with bacteria. UV‐absorbing matrices function as energy mediators that transfer the absorbed photoenergy from an irradiation source to the surrounding sample molecules, resulting in minimum fragmentation. A surprisingly high number of fungal groups have been studied: (i) the terverticillate penicillia, (ii) aflatoxigenic, black and other aspergilli, (iii) Fusarium, (iv) Trichoderma, (iv) wood rotting fungi (e.g. Serpula lacrymans) and (v) dermatophytes. The technique has been suggested for optimizing quality control of fungal Chinese medicines (e.g. Cordyceps). MALDI‐TOF ICMS offers advantages over PCR. The method is now used in taxonomic assessments (e.g. Trichoderma) as distinct from only strain characterization. Low and high molecular mass natural products (e.g. peptaibols) can be analysed. The procedure is rapid and requires minimal pretreatment. However, issues of reproducibility need to be addressed further in terms of strains of species tested and between run variability. More studies into the capabilities of MALDI‐TOF ICMS to identify fungi are required.     

Indoor wood-decay basidiomycetes: damage,causal fungi,physiology, identification and characterization,prevention and control

Indoor wood-decay fungi cause considerable economical damage. Most of the structural damage to the indoors of buildings in Europe and North America is caused by brown-rot fungi that degrade conifer wood; white-rot fungi, which preferentially attack hardwoods, are less common. This review covers the approximately 80 basidiomycetes that commonly occur in buildings. Emphasis was placed on Serpula lacrymans, which is the most common indoor basidiomycete in central Europe. Meruliporia incrassata, the North American pendant to S. lacrymans, has also received considerable attention. In terms of indoor wood decay, moisture and temperature are the most important influences. Wood samples with a low moisture content can be degraded. High temperatures as an alternative control measure do not kill mycelia, with some species surviving in wood samples in the form of heat-resistant arthrospores at temperatures as high 95°C. For refurbishment and scientific purposes, the identity of the causal species should be known. More recently, several molecular techniques have been used to identify fungi; these results are often conflicting with those obtained by other, earlier applied methods. Sequencing of the internal transcribed spacers (ITS) of the rDNA is currently the best molecular tool. Among the other methods available, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) has also been shown to be able to distinguish closely related sister taxa. For further characterization of indoor basidiomycetes, the complete sequences of the 18S, 28S rDNA and the intergenic spacers with the included 5S rDNA have been acquired for some species. If current projects involving whole funal genome sequencing are not taken into account, Antrodia vaillantii is the first basidiomycete for which the complete rDNA sequence has been deposited. The review closes with fundamentals on the prevention and control of indoor wood decay. An erratum to this article can be found at     

Improved molecular methods to characterise Serpula lacrymans and other Basidiomycetes involved in wood decay

Early detection of indoor wood-decay fungi is crucial to prevent building deterioration and thereby avoid considerable economic loss. Due to their increased sensitivity, two reliable DNA-based fingerprinting techniques, capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) and denaturing high-performance liquid chromatography (DHPLC), were used to identify Serpula lacrymans and to profile wood-rot Basidiomycetes in the built environment. Molecular fungal diversity was assessed on 74 environmental samples, collected from 2003 to 2009 from infected buildings in France. S. lacrymans, the most widespread, indoor wood-decay fungus accounted for 64% of total wood-rot Basidiomycetes. A number of other common wood-rot fungi such as Coniophora puteana, Trametes versicolor and Donkioporia expansa were identified. Other Basidiomycetes such as Phlebiopsis gigantea and Scleroderma verrucosum were detected for the first time in the built environment. Reliable diagnostic tools were developed using two PCR-based molecular typing techniques, one for routine diagnosis and another one for community inventories. Together they provided useful data for characterising the complexity of wood-decay ecosystems and helped reveal the coexistence of different wood-decay fungi within the same microbiotope.     

The effect of α-aminoisobutyric acid on wood decay and wood spoilage fungi

The amino acid analogue α-aminoisobutyric acid (AIB) decreased linear extension growth in fifteen out of sixteen wood decay and wood spoilage fungi. In Serpula lacrimans inhibition of extension growth by AIB was accompanied by an increase in the frequency with which the hyphae of the fungus initiated branches. AIB was shown to have a preservative effect against Lentinus lepideus, Serpula lacrimans and Pleurotus ostreatus when wood blocks were impregnated with this chemical prior to challenge by cultures of these fungi. The effectiveness of this compound in limiting growth in a large number of different fungi suggests that competitive inhibitors of nitrogen uptake and metabolism could be used to control fungi which decay wood and similar materials, and may also have wider applications.     

Detection of fungal infestations of wood by ion mobility spectrometry

During their growth on wood many fungi produce characteristic volatile organic compounds as secondary metabolites. These microbial volatile organic compounds (MVOCs) can be used as indicators of fungal growth even when such growth is concealed. In order to investigate the formation of these volatile metabolites on building materials, specimens of pine sapwood on agar media colonized by the dry-rot fungus Serpula lacrymans and a mixture of six moulds were examined. MVOCs from this fungal growth were studied over a period of up to half a year by ion mobility spectrometry (IMS) headspace analysis using a sensitive, portable IMS mini-device. The IMS headspace spectra from the growing fungal specimens obtained during this time span are differed from non-incubated wood specimens and indicate the presence of a mixture of MVOCs. The composition and amount of volatile metabolites of the fungi changed during cultivation. This was confirmed by a principal component analysis (PCA). Identification of different MVOCs in the headspace according to drift time and the mobility of ionized gaseous species in reference to GC-MS investigations were proposed. It was concluded that IMS can be used as a rapid and sensitive on-site method to indicate actively growing fungi concealed within wood.     

Bacterial cell wall-degrading enzymes induce basidiomycete natural product biosynthesis

Natural products play a vital role for intermicrobial interactions. In the basidiomycete arena an important representative is variegatic acid, a lactone natural product pigment whose ecological relevance stems from both inhibiting bacterial swarming and from indirect participation in breakdown of organic matter by brown-rotting fungi. Previous work showed that the presence of bacteria stimulates variegatic acid production. However, the actual external molecular trigger that prompts its biosynthesis in the mushroom hyphae remained unknown. Here, we report on the identification of Bacillus subtilis subtilisin E (AprE) and chitosanase (Csn) as primary inducers of pulvinic acid pigment formation. Using the established co-culture system of B. subtilis and Serpula lacrymans, we used activity-guided FPLC-based fractionation of B. subtilis culture supernatants and subsequent peptide fingerprinting to identify candidates, and their role was corroborated by means of a pigment production assay using heterologously produced chitosanase and subtilisin. B. subtilis mutants defective in either the aprE or the csn gene still triggered pigmentation, yet to a lower degree, which points to a multicausal scenario and suggests the combined activity of these cell wall polymer-attacking enzymes as true stimulus.     

Effects of Supraoptimal Temperatures on Merulius lacrymans

The growth rate of Merulius lacrymans (Jacq.) Fr. decreases very rapidly at temperatures above optimum (about 22°C). No growth occurred above 28°C. The thermal death-time of the fungus at different temperatures above maximum was determined. At 37.5°C the thermal death-time is 4 hours. At this temperature, the respiration is not directly affected, since an appreciable respiration occurred even after 6 hours. On the basis of ¹⁴C-experiments and analysis of UV-absorbing material excreted from the mycelium at supraoptimal temperatures, it is suggested that the detrimental effects of supraoptimal temperatures on this fungus include a degradation of the nucleic acids and a subsequent leakage of the nucleotides out of the cells.     

Genome-wide analysis of simple sequence repeats in the model medicinal mushroom Ganoderma lucidum

Simple sequence repeats (SSRs) or microsatellites are one of the most popular sources of genetic markers and play a significant role in gene function and genome organization. We identified SSRs in the genome of Ganoderma lucidum and analyzed their frequency and distribution in different genomic regions. We also compared the SSRs in G. lucidum with six other Agaricomycetes genomes: Coprinopsis cinerea, Laccaria bicolor, Phanerochaete chrysosporium, Postia placenta, Schizophyllum commune and Serpula lacrymans. Based on our search criteria, the total number of SSRs found ranged from 1206 to 6104 and covered from 0.04% to 0.15% of the fungal genomes. The SSR abundance was not correlated with the genome size, and mono- to tri-nucleotide repeats outnumbered other SSR categories in all of the species examined. In G. lucidum, a repertoire of 2674 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. The highest SSR relative abundance was found in introns (108 SSRs/Mb), followed by intergenic regions (84 SSRs/Mb). A total of 684 SSRs were found in the protein-coding sequences (CDSs) of 588 gene models, with 81.4% of them being tri- or hexa-nucleotides. After scanning for InterPro domains, 280 of these genes were successfully annotated, and 215 of them could be assigned to Gene Ontology (GO) terms. SSRs were also identified in 28 bioactive compound synthesis-related gene models, including one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), three polysaccharide biosynthesis genes and 24 cytochrome P450 monooxygenases (CYPs). Primers were designed for the identified SSR loci, providing the basis for the future development of SSR markers of this medicinal fungus.     

A Review Molecular Typing Methods for <Emphasis Type="Italic">Aspergillus flavus</Emphasis> Isolates

Aspergillus flavus is the second most important Aspergillus species causing human infections. The importance of this fungus increases in regions with a dry and hot climate. Small phylogenetic studies in Aspergillus flavus indicate that the morphological species contains several genetically isolated species. Different genotyping methods have been developed and employed in order to better understand the genetic and epidemiological relationships between environmental and clinical isolates. Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. Typing techniques can also give us a deeper understanding of the colonization pattern in patients. Most of these studies focused on Aspergillus fumigatus because it is medically the most isolated species. To date, there has not been any publication exclusively reviewing the molecular typing techniques for Aspergillus flavus in the literature. This article reviews all these different available methods for this organism.     

Montan wax improves performance of boron-based wood preservatives

Importance of boron compounds in wood preservation is increasing due to their low environmental impact, high efficacy and the fact that many other active ingredients have been removed from the market after the introduction of the Biocidal Products Directive. The most important drawback of boron is prominent leaching in wet environment. In order to improve their fixation, and performance against wood decay fungi, boric acid was combined with montan wax emulsion. Possible synergistic effects of boric acid and montan wax were determined according to modified EN 113 procedure. Norway spruce and beech wood specimens were exposed to three white rot (Trametes versicolor, Pleurotus ostreatus and Hypoxylon fragiforme) and brown rot wood decay fungi (Gloeophyllum trabeum, Antrodia vaillantii and Serpula lacrymans) for 12 weeks. Boron leaching from vacuum/pressure treated Norway spruce wood was determined according to the continuous (EN 84 and ENV 1250-2) and non-continuous (OECD and prCEN/TS 15119-1) procedures. Boron was determined with ICP mass spectrometry in collected leachates. The results of the fungicidal tests clearly showed that montan wax emulsion and boric acid act synergistically against tested wood decay fungi. Approximately 50% lower boric acid retentions are required in combination with montan wax emulsions to achieve sufficient protection against wood rotting fungi. However, it is even more important that all leaching tests performed proved that the addition of montan wax decreased boron leaching from impregnated specimens for 20% up to 50%.     

Preliminary studies on Ramularia crupinae sp. nov. as a potential biological control agent for common crupina (Crupina vulgaris) in the USA

Common crupina, Crupina vulgaris (Asteraceae), is an alien weed of pastures and disturbed noncrop lands in the western USA. Research is underway in Europe to search for the natural enemies to control the weed. Among various natural enemies attacking C vulgaris, a new fungal species, Ramularia crupinae, has been found to cause serious damage to the weed. The fungus has been grown on various media and a modified V8 juice agar has proved to be suitable for both growth and sporulation. Greenhouse and field studies have shown that R. crupinae can kill infected leaves, thus reducing the plant growth, and in severe cases can kill the young plants. During preliminary host-range tests using various members of the Asteraceae, including artichoke and safflower, the fungus attacked only C. vulgaris.     

Pathogenicity of the Entomopathogenic Fungus, <Emphasis Type="Italic">Lecanicillium muscarium</Emphasis>, against the Sweetpotato Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> under Laboratory and Glasshouse Conditions

The potential for using the entomopathogenic fungus Lecanicillium muscarium to control the sweetpotato whitefly, Bemisia tabaci has been established in the laboratory by other studies. Laboratory studies however frequently overestimate the level of control achieved by biological control agents in the glasshouse. Before full-scale commercial or field development is considered, glasshouse trials are required to confirm laboratory results. Under both controlled laboratory and glasshouse conditions high mortality of second instar B. tabaci was recorded after application of L. muscarium. The potential of incorporating L. muscarium into integrated pest management strategies for the control of B. tabaci is discussed.