Assessment of the biocontrol potential of a Trichoderma viride isolate: Part II: Protection against soft rot and basidiomycete decay

A previous paper reported on the establishment of a field and fungal cellar trial set up to determine the biocontrol potential of a specific Trichoderma isolate against wood decay fungi. This paper reports on the analyses used to examine the protective effect of the selected isolate, and presents results indicating an initial protective effect against both basidiomycetes and soft rot fungi. The parameters assessed in the field and fungal cellar trials were soft rot decay, basidiomycete decay, Trichoderma colonisation, moisture content and nitrogen content. The results of these analyses show that the introduction of a biological control agent has had a significant effect on moisture content, decay and nitrogen content. A protective effect has been observed against soft rot and basidiomycete decay fungi in field samples.     

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.     

Diversity and decay ability of basidiomycetes isolated from lodgepole pines killed by the mountain pine beetle

When lodgepole pines (Pinus contorta Douglas ex Louden var. latifolia Engelm. ex S. Watson) that are killed by the mountain pine beetle (Dendroctonus ponderosae) and its fungal associates are not harvested, fungal decay can affect wood and fibre properties. Ophiostomatoids stain sapwood but do not affect the structural properties of wood. In contrast, white or brown decay basidiomycetes degrade wood. We isolated both staining and decay fungi from 300 lodgepole pine trees killed by mountain pine beetle at green, red, and grey stages at 10 sites across British Columbia. We retained 224 basidiomycete isolates that we classified into 34 species using morphological and physiological characteristics and rDNA large subunit sequences. The number of basidiomycete species varied from 4 to 14 species per site. We assessed the ability of these fungi to degrade both pine sapwood and heartwood using the soil jar decay test. The highest wood mass losses for both sapwood and heartwood were measured for the brown rot species Fomitopsis pinicola and the white rot Metulodontia and Ganoderma species. The sap rot species Trichaptum abietinum was more damaging for sapwood than for heartwood. A number of species caused more than 50% wood mass losses after 12 weeks at room temperature, suggesting that beetle-killed trees can rapidly lose market value due to degradation of wood structural components.     

A selective medium for the isolation of wood-rotting basidiomycetes

A selective medium for growing wood-rotting basidiomycetes is described. The medium is based on a mixture of benomyl and 2-phenylphenol which suppresses the growth of lower fungi and Sistotrema brinkmannii, a basidiomycete of frequent occurrence in timber but one which cannot decay wood. Use of the medium in isolation studies permits a higher recovery of wood-rotting basidiomycete fungi than can be achieved by the use of other selective media.     

Inhibition and Stimulation Effects in Communities of Wood Decay Fungi: Exudates from Colonized Wood Influence Growth by Other Species

The effects of exudates from uncolonized and from partly decayed beech wood on the extension rates of 16 later stage decay fungi were investigated. The partly decayed wood had been colonized by the pyrenomycete Eutypa spinosa, or the basidiomycetes Fomes fomentarius, Stereum hirsutum, and Trametes versicolor, all known as common early decay agents in European beech forests. Sterilized wood pieces were placed onto 0.5% malt agar, opposite to small agar plugs containing the test fungi. The latter showed very variable and species-specific growth responses to the various wood types. The presence of uncolonized wood stimulated extension rates in many species, whereas the four previously decayed wood types had variable stimulatory or inhibitory effects. Wood decayed by S. hirsutum resulted in reduced extension rate, delayed growth, or total inhibition in the majority of species, thus it is suggested that this species uses secondary metabolites in a defensive strategy. A single species was, however, stimulated in the presence of S. hirsutum-decayed wood. In contrast, the presence of wood decayed by F. fomentarius was stimulatory to 45% of the species. The other previously decayed wood types generally resulted in more variable responses, depending upon species. The results are discussed in an ecological context and it is suggested that the exudates from the partly decayed wood that are responsible for the reported effects may function as infochemicals, structuring microbial communities in wood.     

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.     

Detection and Identification of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplified Genes Encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

VARIATION IN RIBOSOMAL DNA AMONG BIOLOGICAL SPECIES OF ARMILLARIA,A GENUS OF ROOT-INFECTING FUNGI

Armillaria is a genus of root-infecting fungi composed of several biological species in North America and Europe. To examine relatedness among biological species, ribosomal DNA (rDNA) from one isolate was cloned and rDNAs from 30 isolates were mapped for eight restriction enzymes. The positions of the large (26S) and small (18S) rRNA cistrons were found by Northern hybridizations of total cellular RNA with rDNA subclones and by alignment of maps with conserved restriction sites present in rRNA genes of other fungi. Nine restriction-site and two length polymorphisms were observed. Eight North American (Roman numerals) and five European (species epithets) biological species could be placed in six classes with respect to rDNA maps (rDNA class 1: I and A. ostoyae; class 2: II; class 3: A. borealis; class 4: V, IX, and X; class 5: III, VII, A. lutea, and A. cepistipes; and class 6: VI and A. mellea). Most, but not all, polymorphisms were in intergenic regions.     

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.     

Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

Fungal decomposition of woody debris of Castanopsis sieboldii in a subtropical old-growth forest

Fungi, especially basidiomycetes, are the primary agents of woody debris decomposition in terrestrial forest ecosystems. However, quantitative data regarding the abundance and decay activity of wood-inhabiting fungi are lacking, especially for tropical and subtropical areas. This study demonstrates the dynamics of decay columns of wood-inhabiting fungi within decaying woody debris of Castanopsis sieboldii and the wood decay activities of those fungi in a subtropical natural forest. Among six basidiomycetes and two ascomycetes observed as sporocarps on fallen boles of C. sieboldii, Microporus affinis was most abundantly observed in terms of frequency of sporocarps and as percentage area of decay columns within cross-sections of boles, especially those in the early stages of decomposition. In decay columns of M. affinis, both acid-unhydrolyzable residue (AUR) and holocellulose decayed simultaneously, and wood relative density decreased to 45.8% of that of fresh C. sieboldii wood. A pure culture decay test under laboratory conditions showed that M. affinis was a strong decomposer of AUR and holocellulose. These results suggest that M. affinis has a central role in lignocellulose decomposition of wood of C. sieboldii in the early stages of decomposition.     

An Antarctic Hot Spot for Fungi at Shackleton's Historic Hut on Cape Royds

The historic expedition huts located in the Ross Sea Region of the Antarctic and the thousands of artifacts left behind by the early explorers represent important cultural heritage from the “Heroic Era” of Polar exploration. The hut at Cape Royds built by Ernest Shackleton and members of the 1907–1908 British Antarctic Expedition has survived the extreme Antarctic environment for over 100 years, but recent studies have shown many forms of deterioration are causing serious problems, and microbial degradation is evident in the historic wood. Conservation work to reduce moisture at the hut required removal of fodder, wood, and many different types of organic materials from the stables area on the north side of the structure allowing large numbers of samples to be obtained for these investigations. In addition, wood from historic food storage boxes exposed in a ravine adjacent to the hut were also sampled. Fungi were cultured on several different media, and pure cultures were obtained and identified by sequencing of the internal transcribed spacer region of rDNA. From the 69 cultures of filamentous fungi obtained, the most predominant genera were Cadophora (44%) followed by Thielavia (17%) and Geomyces (15%). Other fungi found included Cladosporium, Chaetomium, and isolates identified as being in Pezizomycotina, Onygenales, Nectriaceae, and others. No filamentous basidiomycetes were found. Phylogenetic analyses of the Cadophora species showed great species diversity present revealing Cadophora malorum, Cadophora luteo-olivacea, Cadophora fastigiata, as well as Cadophora sp. 4E71-1, a C. malorum-like species, and Cadophora sp. 7R16-1, a C. fastigiata-like species. Scanning electron microscopy showed extensive decay was present in the wood samples with type 1 and type 2 forms of soft rot evident in pine and birch wood, respectively. Fungi causing decay in the historic wooden structures and artifacts are of great concern, and this investigation provides insight into the identity and species diversity of fungi found at the site. The historic woods and other organic materials at this site represent a large input of carbon into the Antarctic environment. This as well as nutrient additions from the nearby Adélie penguin (Pygoscelis adeliae) colony and favorable conditions for fungal growth at Cape Royds appear responsible for the significant fungal diversity, and where extensive decay is taking place in wood in contact with the ground.     

Assessment of fungal diversity and deterioration in a wooden structure at New Harbor, Antarctica

Scientists working at New Harbor, Antarctica in November 1959 used a wooden crate as a makeshift workspace and kitchen. The structure has been used intermittently over the subsequent decades and still remains at the site with various materials left in and around it. The wooden structure was assessed for deterioration and samples collected to determine the diversity of fungi at the site after 43 years in the Antarctic environment. Results from these investigations are compared to the results from research on the historic huts of Ross Island, approximately 70 km east of New Harbor that were built 48–58 years earlier. Our analysis shows the wood of the New Harbor structure is extremely weathered and soft rot decay was detected in the wood in contact with the ground. Fungal cultures isolated from wood of the structure were identified using sequences of the internal transcribed spacer region of the rDNA. Several species of Cadophora were identified including C. malorum, C. luteo-olivacea, C. fastigiata and a previously undescribed species designated C. sp. NH. Laboratory decay experiments using two Cadophora species isolated from New Harbor demonstrated extensive decay and loss of biomass in hardwood wafers after 16 weeks. Other fungi isolated from the wood included species of Cladosporium, Hormonema, Penicillium and Lecythophora. Wind erosion has also severely affected the structure’s exterior wood causing deep furrowing between earlywood and latewood cells. In general, the deterioration and fungi found at the site were similar to those found at the historic expedition huts on Ross Island, however, one species obtained is unique to the New Harbor site. This research expands our knowledge of the microbes colonizing wood brought into the polar environment and provides additional information on deterioration and decomposition processes occurring in Antarctica.     

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.     

Molecular detection,community structure and phylogeny of ericoid mycorrhizal fungi

Through traditional culturing and molecular characterization, we have determined that five putative species and 2 polyphyletic assemblages of fungi produce ericoid mycorrhizae in Gaultheria shallon, other Ericaceae and Epacridaceae. Using phylogenetic analysis of ITS2 sequences in GenBank, we have confirmed that most of these fungi occur in North America, Europe, and Australia. The low recovery rate of culturable ericoid mycorrhizal fungi from Gaultheria shallon may partly be explained by the fact that most mycorrhizal root segments contain an unculturable basidiomycete, revealed by direct amplification, cloning, and sequencing of LSU fungal DNA from root. Molecular characterization and phylogenetic analysis are powerful tools in revealing the geographic distribution and identity of ericoid mycorrhizal fungi.     

Ectomycorrhizas of <Emphasis Type="Italic">Coltricia</Emphasis> and <Emphasis Type="Italic">Coltriciella</Emphasis> (Hymenochaetales,Basidiomycota) on Caesalpiniaceae,Dipterocarpaceae and Myrtaceae in Seychelles

Ectomycorrhizal fungi, especially basidiomycetes, have repeatedly evolved from saprotrophic ancestors. Using rDNA internal transcribed spacer and large subunit sequences, we demonstrate that four species of Coltricia and Coltriciella form ectomycorrhiza with the native Vateriopsis seychellarum (Dipterocarpaceae) and Intsia bijuga (Caesalpiniaceae) as well as the introduced Eucalyptus robusta (Myrtaceae) in Seychelles. Coltricia and Coltriciella species share a thin, orange-brown to dark brown mantle and extremely thick, clampless hyphae. Phylogenetic analyses placed Coltriciella monophyletic within Coltricia. This study provides further evidence that fruiting habit on dead wood does not indicate saprotrophic lifestyle.     

Development of microsatellite markers for the red‐listed wood‐decay fungus Phlebia centrifuga

Seven polymorphic microsatellite markers were developed for the wood‐decay basidiomycete Phlebia centrifuga. The primers were identified using two techniques, based on intersimple sequence repeats (ISSR) and amplified fragment length polymorphism (AFLP), respectively. The markers were screened on 27 isolates from Europe and North America. Two markers varied only on a worldwide scale, but not within Europe. The other five showed variation on both scales. These markers will now be used to characterize populations of P. centrifuga, which is red‐listed as near‐threatened in its natural habitat due to human disturbance.     

Diversity of fungi in creosote-treated crosstie wastes and their resistance to polycyclic aromatic hydrocarbons

This study was conducted to generate information regarding the diversity of fungi inhabiting creosote-treated wood in a storage yard for crosstie wastes in Gwangmyeong, Korea. Additionally, the resistance to polycyclic aromatic hydrocarbons (PAHs) of indigenous fungi that mainly occupy creosote-treated wood was evaluated. We isolated fungi from the surface and inner area of crosstie wastes and identified them using a combination of traditional methods and molecular techniques. Overall, 179 isolates including 47 different species were isolated from 240 sampling sites. The identified fungal species included 23 ascomycetes, 19 basidiomycetes, and 5 zygomycetes. Three species, Alternaria alternata, Irpex lacteus, and Rhizomucor variabilis, were the most frequently isolated ascomycetes, basidiomycetes, and zygomycetes, respectively. The results of this study showed that there was a large difference in the fungal diversity between the surface and the inner area. Additionally, zygomycetes and ascomycetes were found to have a greater tolerance to PAHs than basidiomycetes. However, two basidiomycetes, Heterobasidion annosum and Schizophyllum commune, showed very high resistance to PAHs, even in response to the highest concentration (1,000 ppm), which indicates that these species may play a role in the degradation of PAHs.     

Characterization of a strong CCA-treated wood degrader,unknown <Emphasis Type="Italic">Crustoderma</Emphasis> species

In this study, basidiomycete isolates that possessed a strong ability to degrade chromated copper arsenate (CCA)-treated wood were characterized. These fungal isolates, which were collected from CCA-treated pine log wastes, showed no recognizable morphological properties on culture media. Nucleotide sequence analysis of the large subunit rDNA of the isolates revealed that they were one species. Based on the high sequence similarity (>95%) and close phylogenetic relationship with several known species of Crustoderma, the fungal isolates characterized in this study were classified as a Crustoderma sp. In a wood degradation test, Crustoderma isolate KUC8611 produced a remarkably higher weight loss in CCA-treated Pinus radiata (68.7%), Pseudotsuga menziesii (39.7%), and Tsuga heterophylla (38.5%) wood than other evaluated basidiomycete species, including Crustoderma flavescens and Crustoderma corneum. In addition, extracellular enzymes for cellulose and protein degradation were detected when the isolates were cultured in chromogenic media, which supports the finding that isolate KUC8611 is a wood degrader. Furthermore, an in vitro test for metal tolerance revealed that isolate KUC8611 showed strong arsenic tolerance, but that it could not tolerate copper. Finally, isolate KUC8611 produced lower amounts of oxalic acid than copper-tolerant fungi such as Fomitopsis palustris and Antrodia vaillantii. To the best of our knowledge, this is the first study to report the degradation of CCA-treated wood by a Crustoderma species.     

Differences in crystalline cellulose modification due to degradation by brown and white rot fungi

Wood-decaying basidiomycetes are some of the most effective bioconverters of lignocellulose in nature, however the way they alter wood crystalline cellulose on a molecular level is still not well understood. To address this, we examined and compared changes in wood undergoing decay by two species of brown rot fungi, Gloeophyllum trabeum and Meruliporia incrassata, and two species of white rot fungi, Irpex lacteus and Pycnoporus sanguineus, using X-ray diffraction (XRD) and ¹³C solid-state nuclear magnetic resonance (NMR) spectroscopy. The overall percent crystallinity in wood undergoing decay by M. incrassata, G. trabeum, and I. lacteus appeared to decrease according to the stage of decay, while in wood decayed by P. sanguineus the crystallinity was found to increase during some stages of degradation. This result is suggested to be potentially due to the different decay strategies employed by these fungi. The average spacing between the 200 cellulose crystal planes was significantly decreased in wood degraded by brown rot, whereas changes observed in wood degraded by the two white rot fungi examined varied according to the selectivity for lignin. The conclusions were supported by a quantitative analysis of the structural components in the wood before and during decay confirming the distinct differences observed for brown and white rot fungi. The results from this study were consistent with differences in degradation methods previously reported among fungal species, specifically more non-enzymatic degradation in brown rot versus more enzymatic degradation in white rot.