Evidence of fungal decay in petrified legume wood from the Neogene of the Bengal Basin,India

Silicified fossil legume woods of Cynometroxylon Chowdhury & Ghosh collected from the Neogene (late Miocene) sediments of the Bengal Basin, eastern India, exhibit fungal decay seldom found in the fossil record. The wood possesses numerous perforate areas on the surface that seem to be the result of extensive fungal activity. In transverse section, the decayed areas (pockets) appear irregular to ellipsoidal in outline; in longitudinal section these areas of disrupted tissue are somewhat spindle-shaped. Individual pockets are randomly scattered throughout the secondary xylem or are restricted to a narrow zone. The aforesaid patterns of decay in fossil wood show similarities with that of white rot decay commonly produced by higher fungi, specifically basidiomycetes and ascomycetes. The host fossil wood harbors abundant ramifying and septate fungal hyphae with knob like swellings similar to pseudoclamps in basidiomycetes, and three-celled conidia-like reproductive structures. This record expands our current knowledge of wood decaying fungi-host plant interaction in the Neogene tropical forests of Peninsular India.     

Immunocytochemical localization of laccase L1 in wood decayed by Rigidoporus lignosus.

The cellular distribution of laccase L1 during degradation of wood chips by Rigidoporus lignosus, a tropical white rot fungus, was investigated by using anti-laccase L1 polyclonal antisera in conjunction with immunolabeling techniques. The enzyme was localized in the fungal cytoplasm and was associated with the plasmalemma and the fungal cell wall. An extracellular sheath, often observed around fungal cells, often contained laccase molecules. Diffusion of laccase within apparently unaltered wood was seldom observed. The enzyme penetrated all degraded cell walls, from the secondary wall toward the primary wall, including the middle lamella. Xylem cells showing advanced stages of decay were sometimes devoid of significant labeling. These data suggest that the initial attack on wood was not performed by laccase L1 of R. lignosus. Previous alteration of the lignocellulose complex may facilitate the movement of laccase within the wood cell walls. This immunogold study revealed that laccase localization during wood degradation seems limited not in space but in time.     

Immunocytochemical localization of laccase L1 in wood decayed by Rigidoporus lignosus.

The cellular distribution of laccase L1 during degradation of wood chips by Rigidoporus lignosus, a tropical white rot fungus, was investigated by using anti-laccase L1 polyclonal antisera in conjunction with immunolabeling techniques. The enzyme was localized in the fungal cytoplasm and was associated with the plasmalemma and the fungal cell wall. An extracellular sheath, often observed around fungal cells, often contained laccase molecules. Diffusion of laccase within apparently unaltered wood was seldom observed. The enzyme penetrated all degraded cell walls, from the secondary wall toward the primary wall, including the middle lamella. Xylem cells showing advanced stages of decay were sometimes devoid of significant labeling. These data suggest that the initial attack on wood was not performed by laccase L1 of R. lignosus. Previous alteration of the lignocellulose complex may facilitate the movement of laccase within the wood cell walls. This immunogold study revealed that laccase localization during wood degradation seems limited not in space but in time.     

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.     

Intra- and Extracellular Localization of Lignin Peroxidase during the Degradation of Solid Wood and Wood Fragments by Phanerochaete chrysosporium by Using Transmission Electron Microscopy and Immuno-Gold Labeling

The distribution of lignin peroxidase during degradation of both wood and woody fragments by the white rot fungus Phanerochaete chrysosporium was investigated by using anti-lignin peroxidase in conjunction with postembedding transmission electron microscopy and immuno-gold labeling techniques. The enzyme was localized in the peripheral regions of the fungal cell cytoplasm in association with the cell membrane, fungal cell wall, and extracellular slime materials. In solid wood, lignin peroxidase was detected in low concentrations associated with both superficial and degradation zones within secondary cell walls undergoing fungal attack. A similar but much greater level of extracellular peroxidase activity was associated with wood fragments degraded by the fungus grown under liquid culture conditions optimal for production of the enzyme. Efforts to infiltrate degraded wood pieces with high levels of lignin peroxidase showed the enzyme to be restricted to superficial regions of wood decay and to penetrate wood cell walls only where the wall structure had been modified. In this respect the enzyme was able to penetrate characteristic zones of degradation within the secondary walls of fibers to sites of lignin attack. This suggests a possibility for a close substrate-enzyme association during wood cell wall degradation.     

Pre-Quaternary wood decay ‘caught in the act’ by fire – examples of plant-microbe-interactions preserved in charcoal from clastic sediments

The fossil record contains abundant evidence for the activity of microorganisms in the form of characteristic decay structures within fossil plant remains. Despite an abundance of charcoal in many sedimentary environments, there is little published evidence of such decay structures within charcoal from pre-Quaternary clastic deposits. The present contribution presents some examples of pre-Quaternary charcoal from clastic sediments which exhibit pre-charring decay structures, stratigraphically reaching from the Permian up to the Oligocene. Examples include specimens affected by the principle types of wood rot known from modern ecosystems (i.e. brown-rot, white rot and soft-rot) as well as a peculiar decay pattern resembling an atypical type of white-rot, which is only rarely known from modern wood. Theoretically there are different, so far hypothetical, scenarios which could be used to explain the scarcity of published reports on such material. Besides taphonomical biases directly influencing the sedimentary record of charcoal towards material not affected by microbial decay, it is conceivable that the lack of reports of such material from pre-Quaternary clastic deposits represents a, maybe unintentional, bias introduced by scientists working on pre-Quaternary charcoal.     

Screening Wood Decayed by White Rot Fungi for Preferential Lignin Degradation

A screening procedure in which scanning electron microscopy was used indicated that 26 white rot fungi selectively removed lignin from various coniferous and hardwood tree species. Delignified wood from field collections had distinct micromorphological characteristics that were easily differentiated from other types of decay. The middle lamella was degraded, and the cells were separated from one another. Secondary cell wall layers that remained had a fibrillar appearance. Chemical analyses of delignified wood indicated that the cells were composed primarily of cellulose. Only small percentages of lignin and hemicellulose were evident. Delignified wood was not uniformly distributed throughout the decayed wood samples. White-pocket and white-mottled areas of the various decayed wood examined contained delignified cells, but adjacent wood had a nonselective removal of lignin where all cell wall components had been degraded simultaneously. This investigation demonstrates that selective delignification among white rot fungi is more prevalent than previously realized and identifies a large number of fungi for use in studies of preferential lignin degradation.     

Penetrability of White Rot-Degraded Pine Wood by the Lignin Peroxidase of Phanerochaete chrysosporium

The penetration of enzymes into wood cell walls during white rot decay is an open question. A postembedding immunoelectron microscopic technique was the method of choice to answer that question. Infiltration of pine wood specimens with a concentrated culture filtrate greatly improved the labeling density and, thereby, reproducibility. Characterization of the concentrated culture filtrate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) revealed three closely spaced proteins of molecular weights about 42,000 showing immunoreactivity against anti-lignin peroxidase serum. It was shown by immunogold labeling that lignin peroxidase of Phanerochaete chrysosporium is located on the surface of the wood cell wall or within areas of heavy attack. It did not diffuse into undecayed parts of the cell wall. The reasons for preventing lignin peroxidase from penetrating wood cell walls during white rot decay are discussed.     

Characterization of Palo Podrido, a Natural Process of Delignification in Wood

Chemical and morphological changes of incipient to advanced stages of palo podrido, an extensively delignified wood, and other types of white rot decay found in the temperate forests of southern Chile were investigated. Palo podrido is a general term for white rot decay that is either selective or nonselective for the removal of lignin, whereas palo blanco describes the white decayed wood that has advanced stages of delignification. Selective delignification occurs mainly in trunks of Eucryphia cordifolia and Nothofagus dombeyi, which have the lowest lignin content and whose lignins have the largest amount of β-aryl ether bonds and the highest syringyl/guaiacyl ratio of all the native woods included in this study. A Ganoderma species was the main white rot fungus associated with the decay. The structural changes in lignin during the white rot degradation were examined by thioacidolysis, which revealed that the β-aryl ether-linked syringyl units were more specifically degraded than the guaiacyl ones, particularly in the case of selective delignification. Ultrastructural studies showed that the delignification process was diffuse throughout the cell wall. Lignin was first removed from the secondary wall nearest the lumen and then throughout the secondary wall toward the middle lamella. The middle lamella and cell corners were the last areas to be degraded. Black manganese deposits were found in some, but not all, selectively delignified samples. In advanced stages of delignification, almost pure cellulose could be found, although with a reduced degree of polymerization. Cellulolytic enzymes appeared to be responsible for depolymerization. A high brightness and an easy refining capacity were found in an unbleached pulp made from selectively delignified N. dombeyi wood. Its low viscosity, however, resulted in poor resistance properties of the pulp. The last stage of degradation (i.e., decomposition of cellulose-rich secondary wall layers) resulted in a gelatinlike substance. Ultrastructural and chemical analyses of this substance showed the matrix to have no microfibrillar structure characteristic of woody cell walls but to still be rich in glucan.     

Wood decay under the microscope

Many aspects of the interactions between host wood structure and fungal activity can be revealed by high resolution light microscopy, and this technique has provided much of the information discussed here. A wide range of different types of decay can result from permutations of host species, fungal species and conditions within wood. Within this spectrum, three main types are commonly recognised: brown rot, white rot and soft rot. The present review explores parts of the range of variation that each of these encompasses and emphasizes that degradation modes appear to reflect a co-evolutionary adaptation of decay fungi to different wood species or the lignin composition within more primitive and advanced wood cell types. One objective of this review is to provide evidence that the terms brown rot, white rot and soft rot may not be obsolete, but rigid definitions for fungi that are placed into these categories may be less appropriate than thought previously. Detailed knowledge of decomposition processes does not only aid prognosis of decay development in living trees for hazard assessment but also allows the identification of wood decay fungi that can be used for biotechnology processes in the wood industry. In contrast to bacteria or commercial enzymes, hyphae can completely ramify through solid wood. In this review evidence is provided that wood decay fungi can effectively induce permeability changes in gymnospermous heartwood or can be applied to facilitate the identification of tree rings in diffuse porous wood of angiosperms. The specificity of their enzymes and the mild conditions under which degradation proceeds is partly detrimental for trees, but also make wood decay fungi potentially efficient biotechnological tools.     

Comparative study on the delignification of Azadirachta indica (L) Del., wood by Chrysosporium asperatum and Trichoderma harzianum

Structural alterations induced in response to degradation by two white rot Basidiomycetes on the secondary xylem of Azadirachta indica (L) Del., was compared. In vitro decay test was employed to investigate the pattern of delignification of Azadirachta wood by Trichoderma harzianum and Chrysosporium asperatum. Wood samples inoculated with both the strains were analyzed for different periods viz. 30, 60, 90 and 120 days after fungal inoculation. Initially there was no appreciable percent weight loss of the wood blocks but later on (after 60 days) it increased rapidly and was found similar for both the strains (43-46% of wood mass). Samples inoculated with both the strains showed dual pattern of degradation i.e. selective delignification in the initial stage followed by simultaneous rot during advance stage of decay. Separation of the cells due to dissolution of middle lamella was the characteristic feature of both strains but in the advanced stage of decay, formation of erosion troughs were conspicuous in all the cell types. Other features such as cell wall thinning, rounded pit erosion, formation of erosion channels and bore holes were also observed frequently. Initially, fungal invasion started through the vessel lumen, followed by all the cell types of the xylem. From the vessels, mycelia entered into the adjacent rays and parenchyma cells through the pits. In advanced stage, degradation was so pronounced that rays were partially or even completely destroyed while many cells including vessels were either deformed or destroyed due to loss of rigidity of their walls. Structural alterations induced in response to C. asperatum and T. harzianum attack is described in details.     

Use of electron microscopy for aiding our understanding of wood biodegradation

Abstract: The use of electron microscopy (EM) has proved to be an invaluable tool for studying structural aspects of lignocellulose degradation by fungi and bacteria and therefore improving our understanding of wood biodegradation. The present review details the application of conventional (SEM, TEM, STEM), analytical (EM X-ray microanalysis, (EDXA)), and immunogold cytochemical EM procedures in the field and gives specific examples of its use for each of the known important types of microbial wood decay including bacterial (tunnelling and erosion), soft rot (cavity and erosion), brown rot and white rot (simultaneous decay and preferential lignin degraders), and discusses important advances made by adopting the techniques. The more recent use of immunogold cytochemistry for studying microbe and fungal enzyme-wood cell wall interactions and its application for localization of specific wood-degrading (laccase, Mn(II) and lignin peroxidases, and cellulases) and H₂O₂ producing (pyranose oxidase) enzymes in situ during white rot decay are also reviewed, as is the application of EM for studying non-enzymatic wood decay. Methods for labelling and detecting wood components (lignin, hemi- and cellulose) in situ by EDXA and enzyme immunogold cytochemistry are also outlined. The use of EM in wood biodegradation research is rapidly expanding and is seen as an important compliment to biochemical and chemical approaches. The future should see even greater advances in our understanding of wood decay as more advanced and recently developed EM techniques are also exploited.     

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。     

Decay resistance of ash,beech and maple wood modified with N-methylol melamine and a metal complex dye

This study evaluated the decay resistance of ash (Fraxinus excelsior L.), beech (Fagus sylvatica L.), and maple (Acer platanoides L.) wood impregnated by a full cell process with N-methylol melamine (NMM) and combined NMM-metal complex dye (NMM-BS) in aqueous solutions. Basidiomycete decay testing involved incubation with Coniophora puteana (brown rot) and Trametes versicolor (white rot) according to a modified EN 113 (1996) standard, while for the soft rot fungal resistance was evaluated following the standard ENv 807 (2001). NMM and NMM-BS modifications at a WPG range of 7–11% provided decay protection against brown rot resulting in a mass loss less than the required limit (3%). The NMM and NMM-BS modified wood showed increased resistance to white rot decay; however, a higher WPG is needed to prohibit attack from this hardwood specific fungus. The metal-complex dye alone revealed biocidal effects against basidiomycetes. An increased WPG in NMM or NMM-BS had a positive impact against soft rot decay and the lowest mass losses after 32 weeks of exposure were obtained with NMM modification at about 18–21% WPG. NMM modification at this WPG range, however, was not sufficient to protect the wood from soft rot decay. The wood of beech and maple showed slightly higher resistance to all decay types than ash, probably due to the poorer degree of modification of the latter.     

Fungal wood decomposer activities influence community structures of myxomycetes and bryophytes on coarse woody debris

Dead wood is an important habitat for forest organisms, and wood decay fungi are the principal agents determining the dead wood properties that influence the communities of organisms inhabiting dead wood. In this study, we investigated the effects of wood decomposer fungi on the communities of myxomycetes and bryophytes inhabiting decayed logs. On 196 pine logs, 72 species of fungi, 34 species and seven varieties of myxomycetes, and 16 species of bryophytes were identified. Although white rot was the dominant decay type in sapwood and heartwood, brown and soft rots were also prevalent, particularly in sapwood. Moreover, white rot and soft rot were positively and brown rot negatively correlated with wood pH. Ordination analyses clearly showed a succession of cryptogam species during log decomposition and showed significant correlations of communities with the pH, water content, and decay type of wood. These analyses indicate that fungal wood decomposer activities strongly influence the cryptogam communities on dead wood.     

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.     

Wood-decay type and fungal guild dominance across a North American log transplant experiment

We incubated 196 large-diameter aspen (Populus tremuloides), birch (Betula papyrifera), and pine (Pinus taeda) logs on the FACE Wood Decomposition Experiment encompassing eight climatically-distinct forest sites in the United States. We sampled dead wood from these large-diameter logs after 2 to 6 y of decomposition and determined wood rot type as a continuous variable using the lignin loss/density loss ratio (L/D) and assessed wood-rotting fungal guilds using high-throughput amplicon sequencing (HTAS) of the ITS-2 marker. We found L/D values in line with a white rot dominance in all three tree species, with pine having lower L/D values than aspen and birch. Based on HTAS data, white rot fungi were the most abundant and diverse wood-rotting fungal guild, and soft rot fungi were more abundant and diverse than brown rot fungi in logs with low L/D values. For aspen and birch logs, decay type was related to the wood density at sampling. For the pine logs, decay type was associated with the balance between white and brown/soft rot fungi abundance and OTU richness. Our results demonstrate that decay type is governed by biotic and abiotic factors, which vary by tree 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.     

Qualitative and quantitative changes of beech wood degraded by wood-rotting basidiomycetes monitored by Fourier transform infrared spectroscopic methods and multivariate data analysis

Beech wood (Fagus sylvatica L.) veneers were cultivated with white and brown rot fungi for up to 10 weeks. Fungal wood modification was traced with Fourier transform near infrared (FT-NIR) and Fourier transform mid infrared (FT-MIR) methods. Partial least square regression (PLSR) models to predict the total lignin content before and after fungal decay in the range between 17.0% and 26.6% were developed for FT-MIR transmission spectra as well as for FT-NIR reflectance spectra. Weight loss of the decayed samples between 0% and 38.2% could be estimated from the wood surface using individual PLSR models for white rot and brown rot fungi, and from a model including samples subjected to both degradation types.