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.     

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.     

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.     

Decay development in living sapwood of coniferous and deciduous trees inoculated with six wood decay fungi

Development of decay and/or discoloration was assessed in the functional sapwood of one coniferous and three deciduous trees after wounding and artificial inoculation with six wood decay fungi. Living stems of mature Douglas fir, beech, oak, and sycamore trees were wounded in spring 2002 and immediately inoculated with brown, white, and soft rot fungi. Extent of discoloration and decay, wood weight loss, and total phenols in the reaction zone (zone of active response at a dynamic interface between living sapwood and wood colonized by decay fungi) were assessed 16 and 28 months after inoculation.     

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.     

我国杨树溃疡病研究进展

杨树溃疡病主要由真菌侵染所致,该真菌是一类世界性分布的真菌,寄主范围十分广泛,可危害多种林木和果树的树干及果实,引起枯枝、溃疡、流胶和果腐等。另外,还可以引起根腐,导致整株树的枯死。发生病害的林木,用材林材性降低,经济林产量降低,造成严重危害和经济损失。文中对其病害分布、致病机理、抗病机制以及抗病育种等进行了综述。     

Pathogenic wood‐decaying fungi in China

Wood‐decaying fungi on living trees in China were surveyed over the last 12 years. In all, 102 potentially pathogenic Basidiomycetes were found in natural forests, forest plantations, parks and gardens, and among them 20 species were recorded for the first time on living trees in China. The host(s), occurrence, type of damage, type of decay and distribution of each species in China are given. Most of these wood‐destroying fungi are polypores in the Aphyllophorales, and the majority were found in temperate and boreal forests. Of all the species detected, 88 species are known to cause white rot, and 14 cause brown rot; 25 species are considered as common, 44 occasional, and 33 rare.     

Anatomical characterization of decayed wood in standing light red meranti and identification of the fungi isolated from the decayed area

To further our understanding of wood decay in living light red meranti (Shorea smithiana) trees, microscopic characteristics of the cell and cell wall degradations of S. smithiana wood in the presence of the decay fungi, the identity of the causal fungi, and the decay potential and pattern by an isolated fungus were investigated. Cell wall degradations, including cell wall thinning, bore holes formation, rounded pit erosion, and eroded channel opening were clearly observed under light and scanning electron microscopy. In transverse view, many large voids resulting from a coalition of degraded wood tissue appeared in the decayed canker zone. All these observations suggest the well-known simultaneous decay pattern caused by white-rot fungi. By phylogenetic analysis based on the sequences of internal transcribed spacer region of ribosomal DNA, a basidiomycete fungus isolated from the decayed wood was identified as Schizophyllum commune. The degradation caused by this fungus on sound S. smithiana wood in an in situ laboratory decay test was classified as the early stage of simultaneous decay, and showed a similar pattern to that observed in the wood samples naturally decayed.     

Chestnut Red Stain: Identification of the fungi associated with the costly discolouration of Castanea sativa

In Europe, fungal pathogens have reduced the overall productivity of sweet chestnut (Castanea sativa) stands and continue to threaten the economic viability of forestry operations. Chestnut Red Stain (CRS) in north‐eastern Spain, locally referred to as Roig, is capable of decreasing the market value of chestnut timber to the point of rendering chestnut coppices uneconomical. Despite its economic importance, the specific cause of this red discolouration is unknown. With the objective of verifying the presence of fungi within the symptomatic wood, and identifying the fungus or suite of fungi associated with the red stain, wood samples were collected and cultured from 37 stumps found in eight recently harvested stands in the Montseny and Montnegre‐Corredor Natural Parks. To separate the fungi associated with CRS from other species inhabiting the chestnut wood, the origin of each fungal culture was mapped in every stump. The fungi were isolated from cultures and identified by sequencing the ITS region. The results provide insight into the fungal community inhabiting chestnut wood and the potential cause of CRS; nine species were identified including two species known to cause decay in chestnut. One of them, Fistulina hepatica, appears to be a likely candidate for the causal agent of CRS. This is the first study reporting the fungi associated with CRS and opens the door to new epidemiological studies focused on F. hepatica.     

Ursachen und Ausmaß von Stammfäulen in Fichtenbeständen auf verschiedenen Standorten

Summary In 9 Norway spruce stands from different growth regions in Southern Germany, which were provided for clear cutting and where considerable damage by red rot was supposed, on experimental plots all trees before felling were surveyed respecting species, diameter, tree-class and soundness. After felling, the stems were tested for fungus attack, the types of rot (root rot or wound rot) were registered, the degree of the damage was determined and the responsible wood-destroying fungi were identified. In all localities almost the same species of fungi were found, but their quote differed considerably according to the predominating type of rot. At some of the investigated stands spruce was not very resistant to root rot, at others—in spite of good nitrogen nutrition—the diseases only resulted from wounds; rots starting from the roots were seldom found. These differences observed in several stands are discussed. Statistical calculations have shown, that the correlation between the degree of decay on the stump and the height of the rot depends on the site. Therefore an estimate of the amount of rotten wood on account of the extent of decay on the stump, certainly is possible for individual stands, but not in universal view.      

Fungal colonization of coastal Douglas‐fir following mechanical commercial thinning damage

Commercial‐aged Douglas‐fir trees mechanically damaged during commercial thinning activities were investigated to quantify fungal taxa present 14 years after thinning. A total of 235 wood cores from 18 damaged and 18 non‐damaged control trees were obtained. Fungi were isolated on malt extract agar and molecular methods were used to identify 48 taxa. Damaged Douglas‐fir trees were more than twice as likely to contain wood‐inhabiting fungi as undamaged trees (p = 0.07) and contained more than 2.5 times more fungi (p = 0.006). However, typical Douglas‐fir stem‐decay fungi were not isolated. Fungi present were mostly endophytes and no significant differences were found in frequencies of decay/non‐decay soft‐rot fungi between damaged and control trees (p = 0.67). Likewise, no significant differences were found for the trend in number of fungi isolates over radial depth into the stem between damaged and control trees (p = 0.29). Frequency of fungal isolates was non‐linearly related to depth in the tree bole. Significant differences between damaged and control trees (p = 0.03) was influenced by the data in the outermost 3 mm. With the exception of these data, there were no significant differences between damaged and control trees in the radial trend in fungal frequency (p = 0.37). Results of this study suggest that wounding in commercial‐aged Douglas‐fir may not contribute significantly to occurrence and species composition of wood inhabiting fungi over the period of a short‐rotation final harvest. However, damage mitigation is recommended supported by a related study showing vertical discolouration affecting all wounded stems.     

4种木材腐朽菌对白桦木材降解能力的比较

木材腐朽大多数由侵蚀木材的真菌所造成(李坚,2002;池玉杰,2003).这些菌能分泌多种酶,把木材中的纤维素、半纤维素和木质素分解为简单的碳水化合物作为生活的养料(Kirk et al.,1987;Higuchi,1990).由于不同的木腐菌的生理特性同,所分泌的酶及酶的活性各不相同(刘欣等,2008),因此,不同的木腐菌所分解木材的各种成分及相对速度就各不相同(Buswell,1987).     

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     

巨桉林下马勃分子系统发育的关系

采集巨桉林下马勃子实体,培养其菌丝体,提取子实体及菌丝体基因组DNA,进行rDNA-ITS区序列的扩增、克隆和序列测定,并对rDNA-ITS不同区域作序列分析,首次构建马勃的系统发育树.结果表明:野外采集获得马勃子实体l0种,其中成功培养6种,测序结果表明马勃rDNA-ITS区长度在607～766 bp之间,系统发育分析表明硬皮马勃属(Scleroderma)与豆包菌(Pisolithus)亲缘关系较近,秃马勃属(Calvatia)、马勃属(Lycoperdon)及横膜马勃属(Vascellum)之间亲缘关系较近,ITSl-5.8S rDNA-ITS2区可建立马勃类真菌属间系统发育树,ITS2区可用于建立马勃类真菌属内系统发育树,3个待定种硬皮马勃Scleroderma sp.11-1,Scleroderma sp.2-2和Scleroderma sp.5-2为金黄硬皮马勃(S.aurantium)的可能性较大.此研究可为探讨巨桉人工林下外生菌根种类与作用机制、马勃分类系统学及菌丝体的开发研究奠定基础.     

Stem-rot damage and the progress of causal fungi in old-aged Japanese larch trees at the foot of Mt. Fuji

Damage caused by stem-rot and the progress of the causal fungi in old-aged Japanese larch (Larix kaempferi (Lamb.) Carr.) was investigated at the foot of Mt. Fuji. Stem-rot was found in 75% of 108 trees investigated, and volume of rot was 6% of the total wood volume in the forest investigated. Stem-rot damage was much greater than the damage by butt-rot.Stereum sanguinolentum (Alb. and Schw. ex Fr.) Fr. infected larch trees at the greatest incidence (49.4%). However,Porodaedalea chrysoloma (Fr.) Imaz. caused the most volume loss to the trees.S. sanguinolentum infected larch stems mainly through stem wounds, and decay caused by the fungus progressed 9.75×10² cm³/year on average.P. chrysoloma infected larch stems mainly through dead branches and wounds, and the average rate of decay progress for the fungus was 2.74×10³ cm³/year.     

Occurrence and decay patterns of common wood‐decay fungi in hazardous trees felled in the Helsinki City

To improve the management of ageing urban trees, the role of wood‐decay fungi as potential causes of stem breakage was investigated among hazardous trees removed in the Helsinki City area during 2001–2004. The study material comprised 194 trees, and included 76 Tilia spp. trees, 58 Betula spp. and 60 Acer spp. Thirteen species or genera of commonly occurring decay fungi were identified on the basis of fruiting bodies and pure cultures. The occurrence of the fungi was investigated in terms of frequency, visibility and as potential causes for stem breakage. Most hazardous fungi caused extensive horizontal decay in the stem; such fungi were Ganoderma lipsiense on Tilia and Acer, Phellinus igniarius on Acer, Inonotus obliquus and Cerrena unicolor on Betula and Kretzschmaria deusta on Acer, Tilia and Betula. Typically, Rigidoporus populinus was frequently present in weak fork formations on Acer trees. Agaric fungi (Pholiota, Armillaria, Pleurotus and Hypholoma) were frequently recorded but were of minor importance from the point of view of tree breakage hazard.     

Assessment of decay risk of airborne wood-decay fungi II: relation between isolated fungi and decay risk

The relationship between the taxa of airborne fungi and the decay risk was investigated. Airborne fungi in 1,000 l of air were trapped on Japanese cedar disks, and incubated in a damp container kept at 26oC. After 16-week incubation, filamentous fungi grown on the disks were isolated and DNA extracted from each isolate was amplified with the primers ITS4/ITS5. The DNA sequences of the amplified products were determined and compared to the sequence data of GenBank to determine the species or genus according to a BLAST search. This search revealed that the isolate consisted of 5 major taxa, namely Bjerkandera sp., Phanerochaete sp. (A), Phanerochaete sp. (B), Polyporales sp. Polyporus arcularius, and 6 minor ones. Statistical analysis revealed that the major taxa were trapped on the disks in similar weather conditions except for Bjerkandera sp., which was trapped at a cooler temperature. The analysis also proved the disks to which Phanerochaete spp. or Polyporales sp. were attached showed higher mass loss. It is concluded that, under these experimental conditions, related species of Phanerochaete sordida play an important role in increasing the decay risk caused by airborne wood-decay fungi.     

6种木材白腐菌对山杨材木质素分解能力的研究

由于不同的木材腐朽菌的生理特性不同 ,所分泌的酶及酶的活性各不相同 ,因此 ,不同的腐朽菌分解木材的各种成分及相对速度就各不相同 ,而且对于木质纤维基质会有不同的中间代谢产物。本项研究选择了火木层孔菌 (Phelliusigniarius)及另外 5种木材分解能力较强的阔叶树上的白腐菌 :粗毛盖菌 (Funaliagallica)、三色革裥菌 (Lenzitestricolor)、冬拟多孔菌 (Polyporellusbrumalis)、偏肿拟栓菌 (Pseudotrametesgibbosa)和血红密孔菌 (Pyc noporussanguineus) ,研究了它们对山杨木材木质素的分解能力 ,测定了经 6种白腐菌分解一定时期的山杨木材木质素的含量 ,作为木材白腐菌对山杨木材木质素生物降解机制的初步研究 ,旨在为山杨木材生物制浆造纸提供应用基础理论研究 ,同时也可为木质素合理的生物转化为有用的化学品、生物漂白、酶处理防止机械浆的返黄、废水治理、纤维素酶解糖化的微生物前处理等提供相关的借鉴研究 ,以期在生产实践中减轻环境污染并充分利用木质素资源。在无菌的条件下 ,将山杨木片样品分别放入以上 6种白腐菌的平板培养基中受菌侵染 ,一定时间后取出 ,去除木片表面的菌丝体 ,然后分别测定未腐朽材和受菌侵染 4 0d、6 0d、80d和 12 0d时木片样品中木质素的含量 ,分析 6种白腐菌对山杨木     

Fomes annosus (Fr.) Cke. und andere Stammfäulepilze in einem Douglasienbestand,Pseudotsuga menziesii (Mirb.) Franco1

Fomes annosus (Fr.) Cke. and other decay fungi in a Douglas fir stand, Pseudotsuga menziesii (Mirb.) Franco. 40% of the trees in a 40 years old Pseudotsuga menziesii stand showed butt rot. 85 trees were analysed for decay fungi. Fomes annosus, the most frequent fungus, also invaded the sap wood. Factors of the soil favourable to the rot and the possibility of transmission of the most frequent decay fungus, Fomes annosus, from (a) neighbouring spruce stands, (b) from the roots of Scots pine from the previous crop arc discussed. Caniophora puteana was isolated from about 10% of the butt rots. The importance of Calocera viscosa which grew out of the central decay of twelve trees as a decay fungus is still under investigation.     

A comparison between decay patterns of the white‐rot fungus Pleurotus ostreatus in chestnut–leaved oak (Quercus castaneifolia) shows predominantly simultaneous attack both in vivo and in vitro

In this research, we examined decay patterns occurring in Quercus castaneifolia wood under natural conditions compared with controlled decay in vivo. Pleurotus ostreatus‐infected oak wood was obtained from the Sari forests in the north of Iran. The species causing decay was verified as P. ostreatus using rDNA‐ITS sequencing of pure cultures from infected sapwood. In addition to P. ostreatus, two wood‐inhabiting Ascomycota, Trichoderma harzianum and T. lixii, were present. Mass loss in oak sapwood samples exposed to P. ostreatus for 60 days was around 10 per cent. Samples were prepared from both naturally decayed wood and wood decayed under controlled conditions and examined using microscopy. P. ostreatus was found to produce a simultaneous white‐rot decay pattern in both conditions.