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.     

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.     

Resource utilization of wood decomposers: mycelium nuclear phases and host tree species affect wood decomposition by Dacrymycetes

Throughout evolution, wood-decaying fungi have adapted to different woody plants, resulting in wide species diversity. Dacrymycetes, which are brown-rot fungi and include host-recurrent species, are useful for studying fungal adaptation to host trees. When estimating the decay abilities of basidiomycetes, the nuclear phases of the mycelium should be considered, since dikaryons are thought to be more efficient wood-decayers than monokaryons; however, the difference in their physiological performances remains largely untested. In this study, we verified the decay capabilities of dikaryotic and monokaryotic mycelia and tested the hypothesis that the host tree-recurrence of wood-decaying fungi results from their resource utilization in each host wood. The mass loss caused by eight dacrymycetous species from wood of four tree species was investigated in pure cultures. The decomposition ability of dikaryons was greater than that of monokaryons in these experiments. Dikaryotization is expected to raise certain physiological parameters, such as the quantity and variety of wood-decomposing enzymes, thus enhancing the decomposition rate of wood decomposers. The high decomposition ability of dikaryons suggests their superiority over monokaryons to survive under natural conditions. All dacrymycetous strains caused high mass loss from Pinus wood, the main host tree of Dacrymycetes. However, most of the individual tested strains did not cause the greatest mass loss from the wood of their original host group. This result suggested that host-recurrence can be partly explained by resource utilization, but it is likely that other micro-organisms and abiotic factors also affect host-recurrence in the field environment.     

Comparison of the thiol-dependent antioxidant systems in the ectomycorrhizal Laccaria bicolor and the saprotrophic Phanerochaete chrysosporium

Sequencing of the Laccaria bicolor and Phanerochaete chrysosporium genomes, together with the availability of many fungal genomes, allow careful comparison to be made of these two basidiomycetes, which possess a different way of life (either symbiotic or saprophytic), with other fungi. Central to the antioxidant systems are superoxide dismutases, catalases and thiol-dependent peroxidases (Tpx). The two reducing systems (thioredoxin (Trx) and glutathione/glutaredoxin (Grx)) are of particular importance against oxidative insults, both for detoxification, through the regeneration of thiol-peroxidases, and for developmental, physiological and signalling processes. Among those thiol-dependent antioxidant systems, special emphasis is given to the redoxin and methionine sulfoxide reductase (Msr) multigenic families. The genes coding for these enzymes were identified in the L. bicolor and P. chrysosporium genomes, were correctly annotated, and the gene content, organization and distribution were compared with other fungi. Expression of the Laccaria genes was also compiled from microarray data. A complete classification, based essentially on gene structure, on phylogenetic and sequence analysis, and on existing experimental data, was proposed. Comparison of the gene content of fungi from all phyla did not show huge differences for multigenic families in the reactive oxygen species (ROS) detoxification network, although some protein subgroups were absent in some fungi.     

丽江老君山国家公园木腐真菌区系组成与分布特征

木腐真菌是自然界中以木材为主要生长基质的一类大型真菌,通过分解倒死木的木质素、纤维素和半纤维素实现森林生态系统的物质循环,是森林生态系统的重要组成部分。研究木腐真菌的生态分布及其与环境因子的关系对分析其在森林生态系统中的生态功能有重要作用。本文在野外调查的基础上对云南省丽江老君山国家公园的木腐真菌物种组成和生态习性进行了分析,共采集标本196份,经鉴定为68种,隶属于8目21科40属,优势科为多孔菌科、拟层孔菌科和锈革孔菌科,优势属为异担子菌属,其区系组成以北温带成分为最多(38.2%),其次是世界广布成分,表现出明显的北温带特征。从生态习性来看,63种为腐生菌,其中51种造成白色腐朽,12种造成褐色腐朽;5种为外生菌根菌。木腐真菌的物种组成在不同林分中存在明显差异,落叶阔叶林中种类最多,共34种;其次为针阔混交林,有26种;暗针叶林有22种。脆波斯特孔菌与粗毛韧革菌在3种林分类型中均有分布且多度较高,是该地区的优势种。     

Comparative analysis of the ligninolytic potential of basidiomycetes belonging to different taxonomic and ecological groups

Screening of the ligninolytic activity of basidiomycetes from the Komarov Botanical Institute Basidiomycetes Culture Collection (LE-BIN), Russian Academy of Sciences, belonging to different taxonomic and ecological groups was performed. The patterns of the position of taxa of active producers of ligninolytic enzymes in the modern system of fungi were identified. Cluster analysis showed that the group of fungi with the greatest ligninolytic and degradation potential includes representatives of the families Pleurotaceae, Polyporaceae, and Phanerochaetaceae, which perform the first stages of wood decomposition. As a result, species of interest for the further study of their oxidative potential and use in biotechnology were selected.     

古田山国家级自然保护区木腐真菌物种多样性及分布

木腐真菌是微生物的一个重要类群, 主要以倒木为生长基质, 通过产生各种水解酶将倒木的纤维素、木质素和半纤维素分解为小分子物质, 对促进森林生态系统中的营养物质循环发挥着重要的生态功能。于2016年8月在浙江古田山国家级自然保护区开展的木腐真菌野外调查, 利用形态学和DNA序列分析对采集的标本进行了物种鉴定, 并分析了木腐真菌的物种组成和地理成分。在采集的158份标本中鉴定木腐真菌45属92种, 其中白腐真菌78种, 褐腐真菌14种。古田山的木腐真菌物种区系组成中, 热带-亚热带成分比例最高。在158份木腐真菌标本中, 97份标本采自直径大于10 cm的倒木或树桩上, 分属于76个种, 是木腐真菌生长的主要基质大小类型; 48份标本采自直径为2-10 cm的枝干上, 分属38个种; 13份标本采自直径小于2 cm的枝干上, 分属12种。不同腐烂等级倒木上生长的真菌数量和种类差异明显, 其中一级腐烂倒木上采集到9份标本(7种), 二级腐烂倒木上采集到86份标本(45种), 三级腐烂倒木上49份标本(29种), 四级腐烂倒木上14份标本(14种)。结果表明, 林分中倒木直径大小和腐烂程度是影响木腐真菌生长与分布的重要因子。     

Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin.

Wood is the main renewable material on Earth and is largely used as building material and in paper-pulp manufacturing. This review describes the composition of lignocellulosic materials, the different processes by which fungi are able to alter wood, including decay patterns caused by white, brown, and soft-rot fungi, and fungal staining of wood. The chemical, enzymatic, and molecular aspects of the fungal attack of lignin, which represents the key step in wood decay, are also discussed. Modern analytical techniques to investigate fungal degradation and modification of the lignin polymer are reviewed, as are the different oxidative enzymes (oxidoreductases) involved in lignin degradation. These include laccases, high redox potential ligninolytic peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase), and oxidases. Special emphasis is given to the reactions catalyzed, their synergistic action on lignin, and the structural bases for their unique catalytic properties. Broadening our knowledge of lignocellulose biodegradation processes should contribute to better control of wood-decaying fungi, as well as to the development of new biocatalysts of industrial interest based on these organisms and their enzymes.     

木材腐朽菌在森林生态系统中的功能

木材腐朽菌是森林生态系统的重要组成部分,在森林生态系统中起着极为重要的降解还原作用,主要包括担子菌门非褶菌目、子囊菌门盘菌纲和半知菌类的部分真菌,能全部或部分降解木材中的木质素、纤维素和半纤维素,其降解机制有3种：白色腐朽、褐色腐朽和软腐朽．木材腐朽菌与生态系统中其它生物关系密切,为很多昆虫、鸟类提供营养,有些昆虫也能使木腐菌得到传播．保护木材腐朽菌的生物多样性是保护森林生态系统、维护生态系统健康的重要因素．     

丰林国家级自然保护区木腐真菌多样性与寄主倒木的关系

木腐真菌是一类以木材为生长基质的大型真菌, 通过分泌各种水解酶全部或部分降解木材中的木质素、纤维素和半纤维素, 促进森林生态系统的物质循环, 具有重要的生态功能。本研究调查了丰林国家级自然保护区固定样地中木腐真菌的多样性和倒木特征, 并进行了木腐真菌的物种多样性和数量与倒木的种类、数量、腐朽程度、直径大小等的相关性分析。结果显示: 在样地内共采集木腐真菌标本295份, 经鉴定为93种, Shannon多样性指数为3.86, Simpson指数为0.96。相关性分析发现木腐真菌的数量和种类与直径为2-5 cm和5-10 cm的倒木、2级腐烂的倒木和红松倒木均显著相关。样地中优势倒木寄主分别为槭属(Acer)、榛属(Corylus)、云杉属(Picea)和松属(Pinus), 这4类倒木上生长的木腐真菌种类组成具有明显的差异, 槭属和榛属倒木上的共有优势种主要是三色拟迷孔菌(Daedaleopsis tricolor)、云芝(Trametes versicolor)和桦附毛孔菌(Trichaptum pargamenum), 而松属和云杉属的共有优势种主要有白囊耙齿菌(Irpex lacteus)、云芝、冷杉附毛孔菌(Trichaptum abietinum)和褐紫附毛孔菌(T. fuscoviolaceum)。倒木产生真菌子实体的概率研究表明, 同一类寄主倒木上发生木腐真菌子实体的概率在调查面积增加到0.36 ha后趋于一个定值, 松属倒木中仅有10.2%产生真菌子实体, 槭属和云杉属分别是12.9%和13.4%, 榛属最高, 达到53.7%。本研究结果对于预测森林生态系统中木腐真菌的发生具有重要理论意义。     

A reverse chemical ecology approach to explore wood natural durability

The natural durability of wood species, defined as their inherent resistance to wood-destroying agents, is a complex phenomenon depending on many biotic and abiotic factors. Besides the presence of recalcitrant polymers, the presence of compounds with antimicrobial properties is known to be important to explain wood durability. Based on the advancement in our understanding of fungal detoxification systems, a reverse chemical ecology approach was proposed to explore wood natural durability using fungal glutathione transferases. A set of six glutathione transferases from the white-rot Trametes versicolor were used as targets to test wood extracts from seventeen French Guiana neotropical species. Fluorescent thermal shift assays quantified interactions between fungal glutathione transferases and these extracts. From these data, a model combining this approach and wood density significantly predicts the wood natural durability of the species tested previously using long-term soil bed tests. Overall, our findings confirm that detoxification systems could be used to explore the chemical environment encountered by wood-decaying fungi and also wood natural durability.     

大小兴安岭针叶树倒木上木腐真菌的物种多样性

木腐真菌在森林生态系统中具有丰富的物种多样性, 并在倒木的降解过程中发挥重要的生态功能。针叶树是大小兴安岭森林生态系统的优势树种, 因此研究针叶树倒木木腐真菌物种多样性和影响其物种分布的相关环境因子有助于揭示大小兴安岭森林生态系统物质循环的机理。本研究收集了近16年对大小兴安岭地区冷杉属(Abies)、落叶松属(Larix)、云杉属(Picea)和松属(Pinus) 4类针叶树倒木上1,561份木腐真菌标本的采集信息, 统计了物种种类及其腐朽类型, 并选取具有代表性的地点开展木腐真菌群落多样性及其与环境因子的相关性分析。结果显示, 大小兴安岭针叶树倒木木腐真菌有166种, 隶属于70属, 其中白腐真菌有111种, 占所有种类的66.9%, 褐腐真菌为55种, 占所有种类的33.1%。在4类针叶树倒木上均能生长的真菌种类有19种, 占所有种类的11.5%, 其中柔丝干酪孔菌(Oligoporus sericeomollis)是各类倒木上木腐真菌群落中的优势种。大兴安岭地区落叶松属为优势寄主, 其倒木上生长的木腐真菌种类数和个体数在4类倒木中均为最高; 而小兴安岭地区松属倒木上木腐真菌种类数和个体数比其他3类倒木高, 是该地区的优势寄主。对6个代表性地区木腐真菌群落的研究显示, 有11种真菌在6个地区均有分布, 小兴安岭地区木腐真菌多样性普遍高于大兴安岭地区; 聚类分析显示树种比地理位置对木腐真菌物种分布的影响更大。     

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.     

Production of new unsaturated lipids during wood decay by ligninolytic basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

Fungal biodegradation and enzymatic modification of lignin

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H₂O₂-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed.     

Persistence of the gelatinous layer within altered tension-wood fibres of beech degraded by Ustulina deusta

The gelatinous layer (G-layer) of tension-wood fibres in reaction wood of beech showed alterations as a result of the physiological processes involved in the conversion of sapwood into false heartwood or reaction-zone tissue. Using transmitted-light, fluorescence and UV microscopy, polyphenolic compounds were found to infiltrate and encrust the cellulose microfibrils within the G-layer. Experiments with naturally infected and artificially inoculated wood showed that these processes affect the rate and mode of degradation by wood-decaying fungi. Thus, although the ascomycete Ustulina deusta was able to degrade the G-layer from within the lumina of tension-wood fibres in unaltered sapwood, it failed to do so for a prolonged period within false heartwood and reaction zones. In both situations, however, there was some degradation of the underlying secondary wall in the form of erosion troughs which can be attributed to soft rot 'type II', and internal cavity formation typical for 'type I' attack. The present study indicates that not only cell type, but also alterations in the cell wall structure, affect the activity and degradation mode of decay fungi in beech.     

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.     

森林生态系统中木腐真菌群落形成机理及生态功能

在森林生态系统中,枯死木是一个重要的组成部分,为很多生物提供栖息地,有助于养分循环以及碳和水的储存.木材分解是森林生态系统养分循环、土壤形成和碳收支的决定性过程,越来越受到森林生态学家、病理学家和管理者的重视.在此过程中,木腐真菌通过分泌多种酶降解木材主要成分,实现生态系统中的物质循环,具有极为关键和重要的作用.木腐真...