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.     

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe³⁺-reductants. Phenolates were the major compounds with Fe³⁺-reducing activity in both fungi and displayed Fe³⁺-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe³⁺ and H₂O₂ (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum—a model brown rot fungus—other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.     

Comparing lignocellulose physiochemistry after decomposition by brown rot fungi with distinct evolutionary origins

Among wood‐degrading fungi, lineages holding taxa that selectively metabolize carbohydrates without significant lignin removal (brown rot) are polyphyletic, having evolved multiple times from lignin‐removing white rot fungi. Given the qualitative nature of the ‘brown rot’ classifier, we aimed to quantify and compare the temporal sequence of carbohydrate removal among brown rot clades. Lignocellulose deconstruction was compared among fungi using distinct plant substrates (angiosperm, conifer, grass). Specifically, aspen, pine and corn stalk were harvested over a 16‐week time series from microcosms containing Gloeophyllum trabeum, Fomitopsis pinicola, Ossicaulis lignatilis, Fistulina hepatica, Serpula lacrymans, Wolfiporia cocos or Dacryopinax sp. After quantifying plant mass loss, a thorough compositional analysis was complemented by a saccharification test to determine wood cell wall accessibility. Mass loss and accessibility varied depending on fungal decomposer and substrate, and trajectories of loss for hemicellulosic components and cellulose differed among plant tissue types. At any given stage of decomposition, however, lignocellulose accessibility and the fraction remaining of carbohydrates and lignin within a plant tissue type were generally the same, regardless of fungal isolate. This suggests that the sequence of plant component removal at this typical scale of characterization is shared among these brown rot lineages, despite their diverse genomes and secretomes.     

Biodegradation of pentachlorophenol by white rot fungi under ligninolytic and nonligninolytic conditions

The roles of lignin peroxidase, manganese peroxidase, and laccase were investigated in the biodegradation of pentachlorophenol (PCP) by several white rot fungi. The disappearance of pentachlorophenol from cultures of wild type strains,P. chrysosporium, Trametes sp. andPleurotus sp., was observed. The activities of manganese peroxidase and laccase were detected inTiametes sp. andPleurotus sp. cultures. However, the activities of ligninolytic enzymes were not detected inP. chrysosporium cultures. Therefore, our results showed that PCP was degraded under ligninolytic as well as nonligninolytic conditions. Indicating that lignin peroxidase, manganese peroxidase, and laccase are not essential in the biodegradation of PCP by white rot fungi.     

Fungal resistance of rubber wood modified by fatty acid chlorides

Decay resistance of Rubber wood (Hevea brasiliensis) esterified with three fatty acid chlorides (hexanoyl chloride (C6), decanoyl chloride (C10) and tetra-decanoyl chloride (C14)) was evaluated. Unmodified and modified wood samples were exposed to a brown rot (Polyporus meliae) and a white rot (Coriolus versicolor) fungus for 12 weeks. Unmodified rubber wood was severely decayed by P. meliae and C. versicolor, which was indicated by significant weight loss. The rate of decay by brown rot was higher than white rot. Modified wood samples exhibited very good resistant to brown and white-rot fungi. The degree of protection increased with increase in degree of modification. P. meliae, a brown rot fungus, removed structural carbohydrate component in unmodified wood selectively whereas, C. vesicolor showed preference to lignin. The FTIR spectra of modified wood exposed to fungi show no significant changes in relative peak intensities of lignin/carbohydrates indicating effectiveness of chemically modified wood in restricting chemical degradation. Chemical modification occurred more efficiently at carbohydrate portion of the wood. Therefore, it is more effective in retarding decay due to P. meliae.     

Durability of five native Argentine wood species of the genera <Emphasis Type="Italic">Prosopis</Emphasis> and <Emphasis Type="Italic">Acacia</Emphasis> decayed by rot fungi and its relationship with extractive content

The natural durability of four Argentinean species of Prosopis and one of Acacia was evaluated in laboratory tests, according to European standards, using three brown rot and one white rot fungi. These tests were complemented by assessing the wood chemical composition. All the species were from moderately slightly durable to very durable (classes 4–1), and in all cases the heartwood was the most resistant to fungal attack. Chemical extractives content (organic, aqueous, tannic and phenolic) was higher in the heartwood. However, species durability was not related to extractive contents nor with wood density. Instead, it is possible that extractives could contribute to natural durability in different ways, including the effects related to the antioxidant properties of some of them.     

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.     

Progressive changes in cell wall components of Pinus radiata during decay

Progressive changes in solubility characteristics and lignin content of Pinus radiata sapwood were assessed when small blocks were subjected to decay by brown (Gloeophyllum trabeum) and white (Perenniporia tephropora) rot fungi. The brown rot species removed lignin in approximate proportion to weight loss up to 10%; thereafter the amount of lignin altered little. In contrast, the decline in lignin content was near linear for P. tephropora. Increases in solubility (particularly with hot water and dilute alkali), in sugar content and in the acidity of aqueous extracts were recorded in wood blocks decayed to 15–20% weight loss. While these effects were more pronounced in samples decayed by G. trabeum, it appears that with both organisms structural components were degraded faster than the products could be utilised. In this case, cell wall chemistry may not have been a major determinant of weight losses.     

Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Biopulping of lignocellulosic material using different fungal species: a review

Biopulping can be an alternative to the traditional methods of pulping. Biopulping use fungi that are known to be able to degrade wood as well as lignin constituent of wood. Amongst these white rot fungi are the most proficient biodegrader. The fungus is non sporulating and is a selective lignin degrader. It colonizes either on living or dead wood and decomposes all wood polymers including lignin and extractives making it to be extremely potential to be used in biopulping. The process of biopulping reduces the utilization of chemical in pulping industry and help in decreasing the environmental hazard caused by normal pulping. The present review deals with diverse aspects of biopulping and their ecological as well as economic significances.     

Metabolic activities and ultrastructure imaging at late-stage of wood decomposition in interactive brown rot - white rot fungal combinations

Basidiomycota brown rot fungus (Fomitopsis pinicola) and two white rot fungi (Phlebia radiata, Trichaptum abietinum) were cultivated on thin slices of spruce wood individually and in interspecies combinations. Within 12 months, F. pinicola substantially decomposed spruce wood observed as mass loss, also in three-species combinations. However, white rot fungi through hyphal interactions negatively affected the brown-rot indicative iron reduction capacity of F. pinicola. Decay-signature gene expression in mycelial interaction zones indicated suppression of brown rot mechanism but stimulation of enzymatic white-rot lignin attack by P. radiata. Wood ultrastructure imaging showed white-rot dominance in the fungal combinations, whereas destructive brown-rot was evident with F. pinicola alone. Our results confirm the dynamic pattern of enzyme production in fungal combinations, and transition from brown to white rot decomposition metabolism during the late stage of wood decay after one year of interspecific interactions.     

Assessment of saccharification efficacy in the cellulase system of the brown rot fungus Gloeophyllum trabeum

Brown rot fungi uniquely degrade wood by creating modifications thought to aid in the selective removal of polysaccharides by an incomplete cellulase suite. This naturally successful mechanism offers potential for current bioprocessing applications. To test the efficacy of brown rot cellulases, southern yellow pine wood blocks were first degraded by the brown rot fungus Gloeophyllum trabeum for 0, 2, 4, and 6 weeks. Characterization of the pine constituents revealed brown rot decay patterns, with selective polysaccharide removal as lignin compositions increased. G. trabeum liquid and solid state cellulase extracts, as well as a commercial Trichoderma reesei extract (Celluclast 1.5 L), were used to saccharify this pretreated material, using β-glucosidase amendment to remove limitation of cellobiose-to-glucose conversion. Conditions varied according to source and concentration of cellulase extract and to pH (3.0 vs. 4.8). Hydrolysis yields were maximized using solid state G. trabeum extracts at a pH of 4.8. However, the extent of glucose release was low and was not significantly altered when cellulase loading levels were increased threefold. Furthermore, Celluclast 1.5 L continually outperformed G. trabeum cellulase extracts, although extent of glucose release never exceeded 22.0%. Results suggest methodological advances for utilizing crude G. trabeum cellulases and imply that the suboptimal hydrolysis levels obtained with G. trabeum and Celluclast 1.5 L cellulases, even at high loading levels, may be due to brown rot modifications insufficiently distributed throughout the pretreated material.     

A screening method for detecting iron reducing wood-rot fungi

A plate assay using the Fe(II) selective dye, ferrozine, for detecting wood-rot fungi with Fe(III) reductive abilities, was developed. The assay is fast, simple and, in most cases, more sensitive than the corresponding liquid medium test. The brown rot fungi, Gloeophyllum trabeum and Laetiporeus sulphureus, displayed higher iron reductive capabilities than white rot fungi, Trametes versicolor, Ganoderma australe and Ceriporiopsis subvermispora.     

Lignin biodegradation and ligninolytic enzyme studies during biopulping of Acacia mangium wood chips by tropical white rot fungi

White rot fungi are good lignin degraders and have the potential to be used in industry. In the present work, Phellinus sp., Daedalea sp., Trametes versicolor and Pycnoporus coccineus were selected due to their relatively high ligninolytic enzyme activity, and grown on Acacia mangium wood chips under solid state fermentation. Results obtained showed that manganese peroxidase produced is far more compared to lignin peroxidase, suggesting that MnP might be the predominating enzymes causing lignin degradation in Acacia mangium wood chips. Cellulase enzyme assays showed that no significant cellulase activity was detected in the enzyme preparation of T. versicolor and Phellinus sp. This low cellulolytic activity further suggests that these two white rot strains are of more interest in lignin degradation. The results on lignin losses showed 20–30% of lignin breakdown at 60 days of biodegradation. The highest lignin loss was found in Acacia mangium biotreated with T. versicolor after 60 days and recorded 26.9%, corresponding to the percentage of their wood weight loss recorded followed by P. coccineus. In general, lignin degradation was only significant from 20 days onwards. The overall percentage of lignin weight loss was within the range of 1.02–26.90% over the biodegradation periods. Microscopic observations conducted using scanning electron microscope showed that T. versicolor, P. coccineus, Daedalea sp. and Phellinus sp. had caused lignin degradation in Acacia mangium wood chips.     

Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Identification of two mutations that cause defects in the ligninolytic system through an efficient forward genetics in the white‐rot agaricomycete Pleurotus ostreatus

White‐rot fungi play an important role in the global carbon cycle because they are the species that almost exclusively biodegrade wood lignin in nature. Lignin peroxidases (LiPs), manganese peroxidases (MnPs) and versatile peroxidases (VPs) are considered key players in the ligninolytic system. Apart from LiPs, MnPs and VPs, however, only few other factors involved in the ligninolytic system have been investigated using molecular genetics, implying the existence of unidentified elements. By combining classical genetic techniques with next‐generation sequencing technology, they successfully showed an efficient forward genetics approach to identify mutations causing defects in the ligninolytic system of the white‐rot fungus Pleurotus ostreatus. In this study, they identified two genes – chd1 and wtr1 – mutations in which cause an almost complete loss of Mn²⁺‐dependent peroxidase activity. The chd1 gene encodes a putative chromatin modifier, and wtr1 encodes an agaricomycete‐specific protein with a putative DNA‐binding domain. The chd1‐1 mutation and targeted disruption of wtr1 hamper the ability of P. ostreatus to biodegrade wood lignin. Examination of the effects of the aforementioned mutation and disruption on the expression of certain MnP/VP genes suggests that a complex mechanism underlies the ligninolytic system in P. ostreatus.     

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.     

Signature Wood Modifications Reveal Decomposer Community History

Correlating plant litter decay rates with initial tissue traits (e.g. C, N contents) is common practice, but in woody litter, predictive relationships are often weak. Variability in predicting wood decomposition is partially due to territorial competition among fungal decomposers that, in turn, have a range of nutritional strategies (rot types) and consequences on residues. Given this biotic influence, researchers are increasingly using culture-independent tools in an attempt to link variability more directly to decomposer groups. Our goal was to complement these tools by using certain wood modifications as ‘signatures’ that provide more functional information about decomposer dominance than density loss. Specifically, we used dilute alkali solubility (DAS; higher for brown rot) and lignin:density loss (L:D; higher for white rot) to infer rot type (binary) and fungal nutritional mode (gradient), respectively. We first determined strength of pattern among 29 fungi of known rot type by correlating DAS and L:D with mass loss in birch and pine. Having shown robust relationships for both techniques above a density loss threshold, we then demonstrated and resolved two issues relevant to species consortia and field trials, 1) spatial patchiness creating gravimetric bias (density bias), and 2) brown rot imprints prior or subsequent to white rot replacement (legacy effects). Finally, we field-tested our methods in a New Zealand Pinus radiata plantation in a paired-plot comparison. Overall, results validate these low-cost techniques that measure the collective histories of decomposer dominance in wood. The L:D measure also showed clear potential in classifying ‘rot type’ along a spectrum rather than as a traditional binary type (brown versus white rot), as it places the nutritional strategies of wood-degrading fungi on a scale (L:D=0-5, in this case). These information-rich measures of consequence can provide insight into their biological causes, strengthening the links between traits, structure, and function during wood decomposition.     

Symptoms, Wood Lesions and Fungi Associated with Esca in Organic Vineyards in Languedoc-Roussillon (France)

We analysed symptoms, wood lesions and fungi associated with esca in mature organic vineyards in the Languedoc‐Roussillon region. Most previous surveys concerning esca syndrome were conducted in conventionally managed vineyards in other regions. We first found that esca may be present at a very low level in vineyards that were not treated with sodium arsenite. Affected vines displayed three types of symptoms: leaves with interveinal necrosis, wilt of entire branches and these two symptoms in combination. During the 3‐year survey, not all affected vines displayed symptoms every year and the same vine rarely displayed the same symptom or combination of symptoms in successive years. The incidence of esca appeared to be correlated with the percentage of vines that died during the survey but no correlation was found with either mortality before the survey or with the age of the vineyard. Observation of cross‐sections of a total of 210 vines with esca symptoms and isolation of fungi from the wood lesions led to similar results to those obtained in conventionally managed vineyards. Four different lesions were identified: a white rot lesion, a brown lesion in a central position, a brown lesion in a sectorial position and a scattered black spotting in healthy wood. The most frequently observed fungal species were Fomitiporia mediterranea (Fom), Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and Eutypa lata. The white rot lesion caused by Fom was generally accompanied by one or more other lesions in the same vine. Similarly, Fom was generally isolated with one or several other fungi. No relationship was detected between the expression of external symptoms and any specific lesion or fungus. Our study also showed that esca and Eutypa dieback are often superimposed in our region of sampling.     

Comparative and population genomic landscape of Phellinus noxius: A hypervariable fungus causing root rot in trees

The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomic analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood‐decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens and trunk pathogen Porodaedalea pini. Many gene families of lignin‐degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3‐beta‐glucan synthases in P. noxius, which may account for its fast‐growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole‐genome sequencing showed this multinucleate species contains abundant poly‐allelic single nucleotide polymorphisms with atypical allele frequencies. Different patterns of intra‐isolate polymorphism reflect mono‐/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.