Degradation of 2,4-dichlorophenol and pentachlorophenol by two brown rot fungi

Wheat straw cultures of the brown rot fungi Gloeophyllum striatum and G. trabeum degraded 2,4-dichlorophenol and pentachorophenol. Up to 54% and 27% 14CO2, respectively, were liberated from uniformly 14C-labeled substrates within 6 weeks. Under identical conditions Trametes versicolor, a typical white rot species employed as reference, evolved up to 42% and 43% 14CO2 and expressed high activities of laccase, manganese peroxidase, and manganese-independent peroxidase. No such activity could be detected in straw or liquid cultures of Gloeophyllum. Moreover, G. striatum degraded both chlorophenols most efficiently under non-cometabolic conditions, i.e. on a defined mineral medium lacking sources of carbon, nitrogen and phosphate.     

Molecular phylogenetics of the Gloeophyllales and relative ages of clades of Agaricomycotina producing a brown rot

The Gloeophyllales is a recently described order of Agaricomycotina containing a morphologically diverse array of polypores (Gloeophyllum), agarics (Neolentinus, Heliocybe) and resupinate fungi (Veluti-Veluticeps, Boreostereum, Chaetodermella), most of which have been demonstrated to produce a brown-rot mode of wood decay and are found preferentially on coniferous substrates. Multiple phylogenetic studies have included taxa of Gloeophyllales, but none have sampled the order thoroughly, and so far only ribosomal RNA genes have been used. Consequently the limits and higher level placement of the Gloeophyllales are obscure. We obtained sequence data for three protein-coding genes (rpb2, atp6, tef1) and three rRNA regions (nuc-ssu, nuc-lsu, 5.8S) in 19 species of Gloeophyllales representing seven genera and analyzed them together with a diverse set of Agaricomycotina, emphasizing Polyporales. Boreostereum, which is suspected to produce a white rot, is the sister group of the rest of the Gloeophyllales, all of which produce a brown rot. Gloeophyllum contains at least two independent clades, one of which might correspond to the genus Osmoporus. White rot and resupinate fruiting bodies appear to be plesiomorphic in Gloeophyllales. Relaxed molecular clock analyses suggest that the Gloeophyllales arose in the Cretaceous, after the origin of Pinaceae.     

Resistance of parenchyma cells in wood to degradation by brown rot fungi

Wood degradation by two basidiomycetes, Fomitopsis pinicola and Laetiporus sulphureus was studied in one conifer and four broadleaved trees: Picea abies (Norway spruce), Acer pseudoplatanus (sycamore), Betula pendula (birch), Quercus robur (common oak) and Robinia pseudoacacia (robinia). Observations of birefringence under polarized light showed that in all hosts both brown rot fungi affected cells of the early wood before those of the late wood. Degradation of cellulose, as shown by the loss of birefringence, was apparent after 6 weeks in the cell wall of fibres and fibre tracheids, but even after 12 weeks, axial parenchyma showed no signs of degradation. The results indicate that both brown rot fungi cause higher weight losses in hosts (P. abies and B. pendula) with a small amount of parenchyma cells, whereas the lowest weight losses are associated with wood containing a high amount of parenchyma cells (Q. robur and R. pseudoacacia). Resistance of parenchyma cells to degradation by brown rot fungi appears to be related to the cell wall morphology of parenchyma cells and may also reflect a low co-evolutionary adaptation of brown rot fungi to the xylem of broadleaved trees.     

The ultrastructure of the stroma of the brown rot fungi

Summary Most of the hyphae forming the medulla of the stroma of the brown rot fungi are 4–7 in diameter and contain food reserves in large vacuoles and lipid bodies. Some stromatal hyphae have very thick walls and perform a protective function. Smaller hyphae (1–2 in diameter) form a network through the medulla and their structure suggests that they initiate the growth of vegetative hyphae and spores after the stroma has passed through a period of rest.     

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR.

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases.     

Cellulolytic activity of white, brown and gray wood rot fungi

Culture fluids obtained from submerged cultures of white, brown and gray wood rot fungi were assayed for the presence of cellulolytic activity complexes against the model substrated carboxymethylcellulose-Na and Standard Whatman cellulose and natural substrates, i.e. celluloses isolated from pine bark and sawdust. The cellulolytic activity of the examined fungal species was highly differentiated. The use of model and natural substrates allowed determination of the high substrate specificity of the cellulase complexes produced by the fungi. Not all the fungi were found to produce EC 3.2.1.4. endo-1, 4-beta-glucanase under the culture conditions employed. All the fungi were, however, able to produce a complex of EC 3.2.1.4. exo-1, 4-beta-glucanases. All the examined fungi were also able to degrade, although to a varied extent, such higher forms of cellulose as Standard Whatman cellulose or natural celluloses isolated from pine bark and sawdust. Determination of the cellulolytic activity of fungi against the above-mentioned specific natural substrates affords the possibility of their practical use.     

Solid substrate growth of white rot fungi on coffee pulp

The presence of several antiphysiological factors limit the use of coffee pulp in monogastric and ruminant feeding. Twenty six white rot fungi were grown under solid substrate conditions in previously ensiled and pressed coffee pulp without adding additional sources of nitrogen. All grew and wholly covered the surface of the substrate. Six of them produced fruiting bodies. The weight loss interval ranged between 6.7–28.0% dry matter before fructification and from 17.0 to 48.7% after fructification. Some fungi biodegraded about 70, 55 and 47% of the total polyphenols, caffeine and permanganate lignin present in the original substrate.     

Fungal accumulation of metals from building materials during brown rot wood decay

This study analyzes the accumulation and translocation of metal ions in wood during the degradation performed by one strain of each of the three brown rot fungi; Serpula lacrymans, Meruliporia incrassata and Coniophora puteana. These fungi species are inhabitants of the built environment where the prevention and understanding of fungal decay is of high priority. This study focuses on the influence of various building materials in relation to fungal growth and metal uptake. Changes in the concentration of iron, manganese, calcium and copper ions in the decayed wood were analyzed by induced coupled plasma spectroscopy and related to wood weight loss and oxalic acid accumulation. Metal transport into the fungal inoculated wood was found to be dependent on the individual strain/species. The S. lacrymans strain caused a significant increase in total iron whereas the concentration of copper ions in the wood appeared decreased after 10 weeks of decay. Wood inoculated with the M. incrassata isolate showed the contrary tendency with high copper accumulation and low iron increase despite similar weight losses for the two strains. However, significantly lower oxalic acid accumulation was recorded in M. incrassata degraded wood. The addition of a building material resulted in increased weight loss in wood degraded by C. puteana in the soil-block test; however, this could not be directly linked specifically to the accumulation of any of the four metals recorded. The accumulation of oxalic acid seemed to influence the iron uptake. The study assessing the influence of the presence of soil and glass in the soil-block test revealed that soil contributed the majority of the metals for uptake by the fungi and contributed to increased weight loss. The varying uptake observed among the three brown rot fungi strains toward the four metals analyzed may be related to the specific non-enzymatic and enzymatic properties including bio-chelators employed by each of the species during wood decay.     

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.     

Fungal hydroquinones contribute to brown rot of wood

The fungi that cause brown rot of wood initiate lignocellulose breakdown with an extracellular Fenton system in which Fe(2+) and H(2)O(2) react to produce hydroxyl radicals (.OH), which then oxidize and cleave the wood holocellulose. One such fungus, Gloeophyllum trabeum, drives Fenton chemistry on defined media by reducing Fe(3+) and O(2) with two extracellular hydroquinones, 2,5-dimethoxyhydroquinone (2,5-DMHQ) and 4,5-dimethoxycatechol (4,5-DMC). However, it has never been shown that the hydroquinones contribute to brown rot of wood. We grew G. trabeum on spruce blocks and found that 2,5-DMHQ and 4,5-DMC were each present in the aqueous phase at concentrations near 20 microM after 1 week. We determined rate constants for the reactions of 2,5-DMHQ and 4,5-DMC with the Fe(3+)-oxalate complexes that predominate in wood undergoing brown rot, finding them to be 43 l mol(-1) s(-1) and 65 l mol(-1) s(-1) respectively. Using these values, we estimated that the average amount of hydroquinone-driven .OH production during the first week of decay was 11.5 micromol g(-1) dry weight of wood. Viscometry of the degraded wood holocellulose coupled with computer modelling showed that a number of the same general magnitude, 41.2 micromol oxidations per gram, was required to account for the depolymerization that occurred in the first week. Moreover, the decrease in holocellulose viscosity was correlated with the measured concentrations of hydroquinones. Therefore, hydroquinone-driven Fenton chemistry is one component of the biodegradative arsenal that G. trabeum expresses on wood.     

Preferential degradation of phenolic lignin units by two white rot fungi.

The differential biodegradation of phenolic and nonphenolic (C-4-etherified) lignin units in wheat straw treated with the white rot fungi Pleurotus eryngii and Phanerochaete chrysosporium was investigated under solid-state fermentation conditions. Two analytical techniques applied to permethylated straw were used for this purpose, i.e., alkaline CuO degradation and analytical pyrolysis (both followed by gas chromatography-mass spectrometry for product identification). Despite differences in the enzymatic machinery produced, both ligninolytic fungi caused a significant decrease in the relative amount of phenolic lignin units during the degradation process. Nevertheless, no differences in the biodegradation rates of phenolic and etherified cinnamic acids were observed. Changes in lignin composition and cinnamic acid content were also analyzed in the phenolic and nonphenolic lignin moieties. The results obtained are discussed in the context of the enzymatic mechanisms of lignin biodegradation.     

Outcome of interspecific interactions among brown-rot and white-rot wood decay fungi

Abstract Interspecific mycelial interactions among brown-rot fungi resulted in either deadlock or replacement of one fungus by the other. Similarly, most of the brown-rot fungi deadlocked with some or all of the whitre-rot fungi tested, while a few were able to replace some of the white-rot fungi. The results indicate similarities in interspecific mycelial interactions among brown-rot fungi and between brown-rot and white-rot fungi. The results further suggest that some brown-rot fungi are capable of invading and occupying domains within white-rot fungal communities in decaying wood.     

Response of subterranean clover cultivars to root rot fungi

Twelve widely grown cultivars of subterranean clover (Trifolium subterraneum) were screened both under controlled environment conditions for their resistance to five fungi commonly associated with root rot and under field conditions for their resistance to natural root infections. All cultivars showed decreased seedling survival (particularly from Pythium irregulare and Rhizoctonia solani), tap and lateral root rot (particularly from Fusarium avenaceum, P. irregulare, and R. solani) and reduced plant size (particularly from R. solani and P. irregulare). Individual cultivars generally differed in their response to the five pathogens and for any one pathogen there was generally a range of cultivar susceptibilities. Cultivars with the best resistance to individual root pathogens were identified. The results for the five individual pathogens under controlled conditions only showed correlation with field data for some of the parameters compared.     

Enzyme activities and substrate degradation during white rot fungi growth on sugar-cane straw in a solid state fermentation

Two strains of Pleurotus spp., grown in solid state fermentation on sugar-cane straw, degraded the dry matter by 50% after 60 days. The rate of substrate consumption and the dry weight of fruiting bodies decreased in consecutive flushings. Both strains vigorously attacked hemicellulose (80% of total degradation) and lignin (70%). Fruiting bodies were rich in protein and lipids, and had a low content of carbohydrates and ash.     

Evidence for cleavage of lignin by a brown rot basidiomycete

Biodegradation by brown-rot fungi is quantitatively one of the most important fates of lignocellulose in nature. It has long been thought that these basidiomycetes do not degrade lignin significantly, and that their activities on this abundant aromatic biopolymer are limited to minor oxidative modifications. Here we have applied a new technique for the complete solubilization of lignocellulose to show, by one-bond ¹H-¹³C correlation nuclear magnetic resonance spectroscopy, that brown rot of spruce wood by Gloeophyllum trabeum resulted in a marked, non-selective depletion of all intermonomer side-chain linkages in the lignin. The resulting polymer retained most of its original aromatic residues and was probably interconnected by new linkages that lack hydrogens and are consequently invisible in one-bond ¹H-¹³C correlation spectra. Additional work is needed to characterize these linkages, but it is already clear that the aromatic polymer remaining after extensive brown rot is no longer recognizable as lignin.     

Biodegradation of lignocellulose in Bermuda grass by white rot fungi analyzed by solid-state 13C nuclear magnetic resonance.

Following the solid-state fermentation of Bermuda grass by two lignin-degrading white rot fungi, compositional changes have been observed in situ by utilization of cross-polarization and magic angle spinning 13C nuclear magnetic resonance difference spectra and interrupted decoupling spectra. Intensity differences in the 13C resonances assigned to specific components of the cell wall were used to observe these changes. Bermuda grass treated with Phanerochaete chrysosporium K-3 exhibited losses primarily in the polysaccharide components, with a smaller proportion of phenolic components also being degraded. In contrast, Ceriporiopsis subvermispora FP 90031-sp removed a proportionate amount of phenolic components compared with polysaccharide components. The results also indicated that C. subvermispora preferentially removes guaiacyl phenolic components relative to syringyl phenolic components, while P. chrysosporium was nonspecific in its attack on phenolic components.     

Pink root rot,a revised name of brown root rot of gentian,and the causal fungi,Pyrenochaeta gentianicola sp. nov. andP. terrestris in Japan

Cultivated gentian (Gentiana scabra var.buergeri) has been seriously damaged by pink root rot since the 1970s. Diseased plants finally collapse after wilting and stunting. More than 40 fungal genera were found to be associated with roots of mature and immature diseased plants or seeds. Among these fungi,Pyrenochaeta gentianicola sp. nov. andP. terrestris were almost always associated with the diseased plants. Their morphologies and temperature responses were compared, and their pathogenicity was also demonstrated by artificial inoculation tests.Part of this work was conducted at the National Institute of Agricultural Sciences (presently National Institute of Agro-Environmental Sciences), Tsukuba S.C., Ibaraki 305, Japan by T. Watanabe.     

Characteristics of Gloeophyllum trabeum alcohol oxidase, an extracellular source of H2O2 in brown rot decay of wood

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (K(m) = 2.3 mM, k(cat) = 15.6 s(-1)). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H(2)O(2), a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.