Diversity and community structure of wood-inhabiting fungi found in Japanese wooden houses analyzed by the next-generation sequencing

The diversity and community structures of wood-inhabiting fungi in 16 decayed wood samples from ten wooden houses in Japan were analyzed using a next-generation sequencing (NGS) to determine the fungi responsible for wood decay. DNA of fungi in decayed wood was extracted directly, the internal transcribed spacer (ITS) region in ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR), and then, sequences of tagged ITS fragments were analyzed by NGS. Results of sequencing indicated that 68 species of ascomycetes, 37 species of basidiomycetes, and one fungus each from Mortierellales and Mucoromycetes were detected. The fungal community structures showed diversity and included various species of ascomycetes. A microscopic examination of cell wall structure in decayed wood samples suggested that some ascomycetes were soft-rot fungi. Heat map analysis indicated that the similarity in the structures of fungal communities was influenced to a greater extent by the wood species of samples than where they were used as a component.     

Qualitative and quantitative PCR methods using species-specific primer for detection and identification of wood rot fungi

Species-specific oligonucleotide primers for detecting wood rot fungi, Gloeophyllum trabeum, Trametes versicolor, Coniophora puteana, and Serpula lacrymans, and the primer detecting a group of related fungi to G. sepiarium were developed. These primer sequences were picked up from the internal transcribed spacer region between small-subunit rDNA and large-subunit rDNA. The species selectivities of the developed primers were checked. Real-time polymerase chain reaction (PCR) was carried out using these highly specific primers to quantitatively detect at least of 0.01 ng genome DNA of the target species. This quantitative PCR was also used to differentiate the target species DNA from mixed species DNA. A PCR-based technique using the species-specific primers would be applicable to multiple-sample assay in diagnosis of wood decay and to investigation of environmental fungal populations. Part of this article was presented at the International Symposium on Wood Science and Technology (IAWPC 2005), Yokohama, November 2005     

Rapid and accurate detection of Ceratocystis fagacearum from stained wood and soil by nested and real‐time PCR

A nested and real‐time PCR assay was developed for the rapid and accurate detection of Ceratocystis fagacearum, which is the causal agent of oak wilt in stained wood and soil. Based on the differences of the internal transcribed spacer (ITS) sequences of Ceratocystis spp., one pair of species‐specific primers, CF01/CF02, was designed. Whereas a 280‐bp product was amplified using the purified DNA from three isolates of C. fagacearum as the template, no PCR product was obtained from template of other 18 fungi. The detection sensitivity was 10 pg genomic DNA per 25‐μl PCR reaction volume. To increase detection sensitivity, a nested PCR was developed by using ITS1/ITS4 as the first‐round primers and CF01/CF02 in the second round, as it can detect 1 pg genomic DNA per 25‐μl PCR reaction volume. More importantly, CF01/CF02 primers were successfully adapted to real‐time PCR with a detection limit of 0.1 pg genomic DNA per 20‐μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood and soil.     

Wood‐rotting basidiomycetes are a minor component of fungal communities associated with Acacia hybrid trees grown for sawlogs in South Vietnam

Acacia hybrid (Acacia mangium × Acacia auriculiformis) clones are widely planted in Vietnam with a total of approximately 400,000 ha to meet the demand for pulpwood, sawn timber and wood chip exports. Silvicultural techniques such as pruning and thinning have been applied to improve productivity and sawlog quality of Acacia hybrid plantations. However, those techniques may also create opportunities for wood decay fungi to enter the Acacia hybrid stems through wounds and cause stem defects that reduce sawlog quality and the value of the plantation. The presence of fungal decay agents in Acacia hybrid trees was examined in two Vietnamese plantations. In July 2011, just prior to a second thinning, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Phan Truong Hai for the isolation of fungi. In July 2012, approximately 18 months after pruning and thinning treatments, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Nghia Trung for the isolation of fungi. DNA sequencing of the rDNA ITS identified the isolates. In May 2015, approximately 4 years after thinning and fertilizer treatments, discoloured and decayed wood samples were taken from the above (7‐year‐old) Acacia hybrid plantation at Phan Truong Hai for fungal identification. DNA was extracted directly from discoloured and decayed wood samples and fungal rDNA ITS amplicons sequenced on a Roche 454 sequencer. The results showed that silvicultural treatments did not affect the fungal communities associated with discoloured and decayed wood of Acacia hybrid plantation at Phan Truong Hai. A total of 135 fungal species or OTUs (operational taxonomic units) were identified, including 82 members of Ascomycota and 52 Basidiomycota.     

Detection of intra- and interspecific variation of the dry rot fungus <Emphasis Type="Italic">Serpula lacrymans</Emphasis> by PCR-RFLP and RAPD analysis

We investigated a genotype-based assay to discriminate the dry rot fungi Serpula lacrymans. DNAs were extracted from 74 isolates from the northern half of Japan, and internal transcribed spacers (ITS) were amplified by polymerase chain reaction. Genotypes of isolates were checked by restriction fragment length polymorphism (RFLP) using two enzymes, Taq I and Hha I. Among the 74 isolates identified as S. lacrymans in terms of morphologic features, 5 isolates were shown to have been misidentified. Random amplified polymorphic DNA (RAPD) analysis was conducted in order to detect the intraspecific diversity of S. lacrymans isolated in Japan. Because no relation between geographical origin and genetic distances was observed, the intraspecific diversity of S. lacrymans is suggested to be small.Part of this report was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April 2002     

Sequencing and quantifying plastid DNA fragments stored in sapwood and heartwood of <Emphasis Type="Italic">Torreya nucifera</Emphasis>

The selection of wood species and the styles of sculpture play key roles in the characterization of Buddhist statues. After Jianzhen, a Chinese Buddhist monk, visited Japan in the mid-eighth century, wood of the genus Torreya had been frequently used to produce single-bole statues. Establishing measures for the accurate identification of wood in the genus Torreya is effective for investigating the drastic change in the production of statues during this period. Analyzing the plastid deoxyribonucleic acid (DNA) fragments extracted from wood is considered helpful in the identification of species in the same genus. This study analyzed the sequences and residual amounts of plastid DNA fragments in the wood of Torreya nucifera. Nucleotide substitutions in the plastid DNA were clearly identified between T. nucifera and the species distributed in China, indicating that the wood of Torreya sp. can be discriminated based on the plastid DNA sequences. DNA polymorphism analyses revealed sequence diversity for the intergenic spacers on the T. nucifera plastid DNA. A series of polymerase chain reaction (PCR) analyses demonstrated that the plastid DNA fragments with a length of approximately 100 bp could be amplified from the residual DNA extracted from the T. nucifera sapwood with longer elapsed years after cutting. Therefore, an identification of wood species in the genus Torreya based on their plastid DNA is considered to be one of the most effective measures taken in the study regarding the historical changes of Buddhist statues.     

Differentiating the two closely related species,Phellinus weirii and P. sulphurascens

Phellinus weirii s.l., an aggressive root rot pathogen, causes extensive wood losses and lowers the productivity of western red cedar (WRC, Thuja plicata), Douglas fir (Pseudotsuga menziesii) and other conifers. This fungus has been recognized as a cedar form (P. weirii s.s.) and a non‐cedar form (P. sulphurascens). Differentiating the two species is difficult because their fruiting bodies and cultural morphologies are very similar. However, differences in growth rate and colony morphology were observed when they were grown on malt extract agar with WRC feeder strips. In addition, different restriction fragment length polymorphism patterns were obtained using (i) the internal transcribed spacer (ITS) region cut with the restriction enzyme RsaI, and (ii) the partial large subunit ribosomal DNA region cut with AgeI and NciI. Furthermore, a new specific primer set was designed from the ITS region of P. weirii s.s. and was used to differentiate it from P. sulphurascens and other decay fungi that are frequently found in coniferous trees. These species‐specific primers will facilitate the detection of P. weirii in standing trees well before visible signs of infection are apparent.     

Detection of Chalara fraxinea in common ash (Fraxinus excelsior) using real time PCR

A real time PCR assay was developed for the detection of Chalara fraxinea in common ash. PCR primers and Taqman probes, based on the internal transcribed spacer region of the multi‐copy gene rDNA, were tested for specificity and sensitivity. The primers amplified an 81 bp fragment for C. fraxinea but did not amplify DNA from other Chalara species or from other fungi isolated from ash, whether pathogenic or saprophytic. The limit of detection was 5 pg of genomic DNA per PCR. Moreover, naturally‐infected samples were correctly diagnosed. A procedure for DNA extraction from woody tissues using an electric drill yielded DNA of an appropriate quality for real time PCR. This molecular method could be useful for routine analysis of this emergent pathogen and for epidemiological studies.     

Fungal colonisation of outside weathered modified wood

Specimens of Scots pine sapwood (Pinus sylvestris L.) and beech wood (Fagus sylvatica L.) were treated with an amino-alkyl-functional oligomeric siloxane, a sodium water glass solution and 1,3-dimethylol-4,5-dihydroxyethylene urea (DMDHEU). Treated and untreated wood specimens were exposed outdoors without ground contact. After 9?months of outside exposure, all specimens showed discolouration caused by infestations of mould and staining fungi on the exposed wood surface. Fungi grown on the sample surface were isolated and identified by microscopic technique and sequencing of PCR-amplified DNA from the ITS region. Primarily, an infestation by ascomycetes and related deuteromycetes was found. The most dominant fungi were Trichoderma sp. and Epicoccum sp.. An infestation of Aureobasidium pullulans was only detected on untreated and DMDHEU-treated samples. There were only marginal differences of fungal infestation between the two wood species.     

Identification of Lophodermium seditiosum and L. pinastri in Swedish forest nurseries using species‐specific PCR primers from the ribosomal ITS region

Lophodermium seditiosum is a serious needle pathogen on pine, particularly in nurseries, and there is a need to detect the pathogen during its latent phase. The internal transcribed spacer (ITS) regions of the rDNA of L. seditiosum and L. pinastri were amplified with universal primers and sequenced. Sequence comparisons of the two species allowed the design of species‐specific primers for the ITS regions. The primers were between 18 and 24 bp long with a minimum of 3 bp differences between the species. These primer pairs did not give any amplification of DNA from any other of the examined fungal species or from healthy Pinus sylvestris needles. It was also possible to identify either L. seditiosum or L. pinastri in infected needles with and without signs of infection using these primer pairs. The method was found to be very useful for detection of latent infections of L. seditiosum in P. sylvestris needles in nurseries.     

Microbial population in rotten living body ofSalix matsudana

The microbial population in rotten living body ofSalix matsudana caused byTrametes suaveolens (L.) was researched. 11 bacteria species (1 species ofBacillus, 2 species ofClostridium and 8 species of non-brood-cell bacteria), 1 species ofActinomyces that belongs to Lavendulac, 8 species of fungi and 6 species ofTrichoderma were isolated from rotten trunk. The hyphae ofTrametes suaveolens mainly existed between rotten sections and discoloration sections. In over-rotten section and healthy section the fungi (Trametes suaveolens) were not isolated. The microbes that lived in the discoloration section were the most in kinds and number and they were the pioneer microbes of wood rotting. Only after they dwelled in wood and eliminated its rot-resistance, could wood-rotting fungi invade wood and caused wood-rotting. Responsible editor: Zhu Hong     

Molecular diagnosis of Phytophthora lateralis in trees,water, and foliage baits using multiplex polymerase chain reaction

A polymerase chain reaction (PCR)‐based protocol for detection of Phytophthora lateralis in plant tissues and water is described. Base‐pair (bp) deletions in both of the ribosomal DNA internal transcribed spacer (ITS) regions in P. lateralis were used to design complementary PCR primer sequences that amplify a 738 bp fragment only if P. lateralis DNA is present in the sample. Universal control primers based on conserved sequences of the nuclear ribosomal small subunit are included in a multiplexed reaction, providing an internal check on the procedure. The universal primers amplify an approximately 550 bp fragment that is common to plants, protists, and true fungi. The procedure reliably detects P. lateralis in cedar stem tissues and in roots. Positive reactions were obtained with as few as 200 P. lateralis zoospores in water.     

Records of Bursaphelenchus spp. (Nematoda,Parasitaphelenchidae) in coniferous timber imported from the Asian part of Russia

Coniferous wood imported from the Asian part of Russia was surveyed in Germany (Mukran ferry terminal, highway and railway border station in Frankfurt/Oder) and Austria (railway in Marchegg, Retz and Wr. Neustadt, Lower Austria). The consignments consisted of mixed timber of Pinus/Picea or Pinus/Larix. Out of 625 samples investigated, 51 samples (8.5%) yielded eight Bursaphelenchus species. Bursaphelenchus mucronatus was found 42 times in Pinus, Picea and Larix wood, Bursaphelenchus hylobianum and Bursaphelenchus fraudulentus were detected twice, in Pinus/Larix and in Larix, respectively. The following species were each found once: Bursaphelenchus leoni in mixed timber of Pinus/Picea, Bursaphelenchus ‘borealis’ in Pinus, Bursaphelenchus hellenicus and Bursaphelenchus paracorneolus in Larix. One sample of Larix wood from Krasnoyarsk region contained a few specimens corresponding to Bursaphelenchus xylophilus. Bursaphelenchus mucronatus was present in about 30% of the samples showing signs of insect attack. A Monochamus species was found in a sample from Irkutsk. Most of the B. mucronatus isolates found belonged to the European genotype, whereas the East Asian genotype was found in three instances. This is the first report of B. fraudulentus, B. hellenicus, B. leoni, B. paracorneolus and the East Asian genotype of B. mucronatus in Russia. Bursaphelenchus hylobianum is the only species found in Russian wood and not in Europe so far. The species were identified morphologically and by internal transcribed spacer (ITS)‐restriction fragment length polymorphism (RFLP) technique. Species‐specific ITS‐RFLP patterns were established for B. hylobianum. In the case of the isolate morphologically corresponding to B. xylophilus, DNA extraction from the available low number of specimens failed to yield sufficient rDNA for ITS‐RFLP analysis.     

Productivity of temperate broad-leaved forest stands differing in tree species diversity

? Understanding the effects of tree species diversity on biomass and production of forests is fundamental for carbon sequestration strategies, particularly in the perspective of the current climate change. However, the diversity-productivity relationship in old-growth forests is not well understood.

? We quantified biomass and above-ground production in nine forest stands with increasing tree species diversity from monocultures of beech to stands consisting of up to five deciduous tree species (Fagus sylvatica, Fraxinus excelsior, Tilia spp., Carpinus betulus, Acer spp.) to examine (a) if mixed stands are more productive than monospecific stands, (b) how tree species differ in the productivity of stem wood, leaves and fruits, and (c) if beech productivity increases with tree diversity due to lower intraspecific competition and complementary resource use.

? Total above-ground biomass and wood production decreased with increasing tree species diversity. In Fagus and Fraxinus, the basal area-related wood productivity exceeded those of the co-occurring tree species, while Tilia had the highest leaf productivity. Fagus trees showed no elevated production per basal area in the mixed stands.

? We found no evidence of complementary resource use associated with biomass production. We conclude that above-ground productivity of old-growth temperate deciduous forests depend more on tree species-specific traits than on tree diversity itself.

     

Molecular identification of decay fungi in the wood of urban trees

Trees are valuable for urban areas, however, are also susceptible to wood rot fungi. For accurate and fast assessment of the severity and evolution of decay in standing trees, a molecular technique was used to identify the causal agents of wood rot. Fruit bodies of wood decay fungi were collected from infected trees in various stands in Germany. Thirty-six species were identified by traditional methods. The DNA of fruit bodies was extracted, ITS-rDNA amplified by PCR, and ITS regions sequenced. Wood samples from infected urban trees were collected, the entire DNA extracted from affected wood parts, and fungal ITS amplified and sequenced. Fungal species were identified by comparing sequence data with the fruit body data. The technique enables an accurate and rapid identification of causal rot fungi in urban trees.     

真菌ITS区序列结构及其应用

分析了真核生物以及原核生物核糖体基因内部转录间隔区序列结构.核rDNA的内转录间隔区ITS序列包含被5.8S rDNA所分隔的ITS1和ITS2两个片段.列举了根据真菌ITS区序列设计的扩增通用引物与特异引物.在此基础上,综述了真菌ITS序列结构在种间亲缘关系研究、植物病原真菌的检测、未知病原真菌的鉴定、近似种或疑难种的确定等方面的应用.     

Phylogeographic patterns of Armillariaostoyae in the western United States

Nuclear ribosomal DNA regions (i.e. large subunit, internal transcribed spacer, 5.8S and intergenic spacer) were sequenced using a direct‐polymerase chain reaction method from Armillaria ostoyae genets collected from the western USA. Many of the A. ostoyae genets contained heterogeneity among rDNA repeats, indicating intragenomic variation and likely intraspecific hybridization. Intragenomic variation was verified by visually editing base‐sequence offsets in regions with insertions/deletions, and using sequence‐specific internal primers to resequence heterogeneous regions. Phylogenetic analyses with Bayesian Inference methods were used to define groups within A. ostoyae. Analysis of A. ostoyae from outside the western USA indicated the presence of a Circumboreal group of A. ostoyae that also occurs in Utah; two other phylogeographic groups were associated with the Rocky Mountain and Pacific Northwest regions of the USA. Mixed sequence types, an indication of intraspecific hybrids, were common in some geographic regions. Hybridization events may have influenced species evolution, contributing to variation in pathogenicity and virulence. The occurrence of these groups and intraspecific hybrids also indicates that paleogeography and paleoclimate may have influenced the phylogeography of A. ostoyae. In addition, other Armillaria species were examined for evolutionary relationships with the groups of A. ostoyae. These findings will provide a basis for future research relating ecological function to genetic diversity within A. ostoyae.     

Identification of the genus Armillaria by specific amplification of an rDNA-ITS fragment and evaluation of genetic variation within A. ostoyae by rDNA-RFLP and RAPD analysis

Genetic variation among Armillaria ostoyae isolates was studied by rDNA-restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. A total of 20 A. ostoyae isolates, mainly obtained from Picea spp. of different geographical origins, were examined. Southern hybridization of whole-cell DNAs digested with AvaII and probed with biotin-labelled cloned rDNA from Saccharomyces carlsbergensis allowed the differentiation of five RFLP groups. UPGMA cluster analysis of RAPD profiles (138 scorable bands) generated by 10 decamer primers (OPA 01-OPA 10) grouped the isolates in subclusters at similarity levels between 40% and 96%, indicating high intraspecific genetic variability. Some isolates of different geographical origins subgrouped together, suggesting that similar mutational events have occurred independently and that genetic exchange and recombination occurs among the DNAs in natural populations. The potential role of historical and current spread of spruce plants on the genetic variation of A. ostoyae isolates in Europe is discussed. Using the primer pair ARM-1 and ARM-2, an Armillaria-specific ITS-DNA fragment of about 660 bp was obtained. No intraspecific RFLP of this amplicon could be revealed, indicating low genetic variability of this region. The established informative RFLP and RAPD markers and also the Armillaria-specific ITS-DNA fragment may be powerful tools for further epidemiological, phylogenetic and host-pathogen interaction studies with A. ostoyae.