Plant-Polysaccharide-Degrading Enzymes from Basidiomycetes

SUMMARY

Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation.     

An extracellular haem-protein from Coriolus versicolor

A haem-containing protein has been isolated from the growth medium of Coriolus versicolor, a wood-rotting basidiomycete. The polypeptide was identified as a ‘peroxidase-type’ haem protein of MW 53 700, which appeared to be a glycoprotein and had a protoporphyrin IX prosthetic group with a mid-point redox potential of ?121 mV. It also bound carbon monoxide suggesting it may act as an oxidase, and liberated hydroxyl radicals from hydrogen peroxide as measured by its ability to release ethylene from methional.     

Starch gel electrophoresis of certain enzymes from five species of Fomes

The electrophoretic banding patterns of five enzymes (esterase, catechol oxidase, peroxidase, alkaline phosphatase, and acid phosphatase) from eleven isolates of five species of the wood-rotting basidiomycete, Fomes, were subjected to mathematical analysis in order to examine taxonomic relationships. Generally, greater similarity was observed between the banding patterns for isolates of the same species than between those for isolates of different species. The experimental and analytical technique used in this study could be an aid for the determination of taxonomic relationships among these and other fungi.     

Degradation of Coal by the Fungi Polyporus versicolor and Poria monticola

We report that two species of basidiomycete fungi (Polyporus versicolor and Poria monticola) grow in minimal liquid or solid medium when supplemented with crushed lignite coal. The fungi also grow directly on crushed lignite coal. The growth of both fungi was observed qualitatively as the production and extension of hyphae. No fungal growth occurred in minimal agar medium without coal. The fungi degraded solid lignite coal to a black liquid product which never appeared in cultures unless fungi and coal were present together. Apparently, lignite coal can serve as the principal substrate for the growth of the fungi. Infrared analyses of the liquid products of lignite degradation showed both similarities to and differences from the original lignite.     

A new polyphenoloxidase test for distinguishing between wood-rotting fungi

White-rot fungi causing the white decay of wood, secrete polyphenol oxidase type enzymes which catalyze the oxidation of diphenols to quinones. If the substrate also contains amino acids, they can be oxidized secondarily to a red pigment, the intensity of which depends on the activity of oxidation oxoenzymes. This finding was used for devising a quick test to demonstrate the oxidases secreted into the cultivation medium by wood-rotting fungi. As substrates we used hydroquinone and glycine. We tested a total of 89 species and 131 strains of wood-rotting fungi. Simultaneously, we compared the newly devised test with the most common test for the detection of polyphenoloxidases, with the guaiacol test. It follows from the results that the new test, in which the intensity of the red colour was estimated colorimetrically, permits to divide the wood-rotting fungi into two groups, the positively and the negatively reacting ones. This division does not fully coincide with that obtained by means of the guaiacol test.     

Utilization and polymerization of lignosulfonates by wood-rotting fungi

The susceptibility of lignosulfonates to the action of lignin-degrading wood-rotting fungi was studied by submitting commercial lignosulfonate (Peritan Na) and fractions of calcium lignosulfonate of different molecular weights to the action of selected white rot fungi. As shown by gel filtration chromatography and determinations according to the nitroso method, lignosulfonates, even in conditions which did not support fungal growth, underwent strong polymerization when brought in contact with typical, extracellular polyphenol oxidase-producing white-rot fungi. Owing to the polymerization, nitroso determinations showed a seeming decrease of lignosulfonate. Polyporus dichrous, an “atypical” white-rot fungus which does not produce extracellular polyphenol oxidase and hence does not cause polymerization of lignosulfonates, was found to degrade 11% of the lignosulfonate available in a solid malt extract medium during 19 days. Addition of lignosulfonate to a rich synthetic liquid growth medium increased the mycelial yield of several white-rot fungi. Trametes versicolor was able to grow on a calcium lignosulfonate fraction with molecular weight 1350 which served as sole source of carbon and energy, but not on fractions of higher molecular weight. The utilization/polymerization of lignosulfonates was shown to depend on concentration and on the presence of additional utilizable sources of carbon.     

Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor.

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).     

Conservative ecological and evolutionary patterns in liverwort–fungal symbioses

Liverworts, the most ancient group of land plants, form a range of intimate associations with fungi that may be analogous to the mycorrhizas of vascular plants. Most thalloid liverworts contain arbuscular mycorrhizal glomeromycete fungi similar to most vascular plants. In contrast, a range of leafy liverwort genera and one simple thalloid liverwort family (the Aneuraceae) have switched to basidiomycete fungi. These liverwort switches away from glomeromycete fungi may be expected to parallel switches undergone by vascular plants that target diverse lineages of basidiomycete fungi to form ectomycorrhizas. To test this hypothesis, we used a cultivation-independent approach to examine the basidiomycete fungi associated with liverworts in varied worldwide locations by generating fungal DNA sequence data from over 200 field collections of over 30 species. Here we show that eight leafy liverwort genera predominantly and consistently associate with members of the Sebacina vermifera species complex and that Aneuraceae thalloid liverworts associate nearly exclusively with Tulasnella species. Furthermore, within sites where multiple liverwort species co-occur, they almost never share the same fungi. Our analyses reveal a strikingly conservative ecological and evolutionary pattern of liverwort symbioses with basidiomycete fungi that is unlike that of vascular plant mycorrhizas.     

Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor.

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).     

Isolation and characterization of chitinolytic Streptomyces sp. MT7 and its antagonism towards wood-rotting fungi

An actinomycetes isolate of Loktak Lake soil, designated as MT7, was characterized and identified as Streptomyces sp. based on fatty acid methyl ester and 16S ribosomal RNA gene analysis. Streptomyces sp. MT7 showed strong and broad spectrum antagonism towards seven out of eight tested wood-rotting fungi. Strain MT7 secretes three vital fungal cell wall lytic enzymes, i.e. chitinase, β-1,3-glucanase, and protease, and siderophores. Extracellularly produced mycolytic enzymes lost their antifungal activity completely after treatment with proteinase K and heat, indicating that the tested antifungal metabolites are heat-sensitive and proteinaceous in nature. Extracellular fluid (ECF) and its organic solvent extract also exhibited potential antagonism towards the tested wood-rotting fungi. Antifungal metabolites were characterized as polyene in nature. Biocontrol traits like co-production of cell wall lytic enzymes and antifungal secondary metabolites including siderophores by Streptomyces sp. MT7 suggests that it could be employed as a potential biocontrol agent against wood-rotting basidiomycetes.     

Volatile compounds from Pinus silvestris stimulating the growth of wood-rotting fungi

Summary Certain volatile organic acids stimulated the growth of Stereum sanguinolentum when added through the gas phase or to the liquid nutrient medium. Some of the acids were identified in extract of heat-treated wood of pine (Pinus silvestris), which had been shown (Suolahti, 1951) to have a growth-promoting effect on Stereum sanguinolentum and other wood-rotting fungi.The effect of volatile materials from a heat-treated piece of pine-wood and of caproic acid was tested on 13 other wood-rotting fungi. The growth of 11 of them was stimulated by volatiles from pine-wood, only 2 by caproic acid.The morphology of Coniophora cerebella was altered when volatiles from a neutral fraction of pine-wood extract was added through the gas phase.     

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.     

Primary Homothallism-relation to Heterothallism in the Regulation of Sexual Morphogenesis in Sistotrema

The wood-rotting basidiomycete, Sistotrema brinkmannii, is an aggregate of biological species possessing several variations—homothallism, bipolar heterothallism and tetrapolar heterothallism—on the genetic regulation of a critical phase of development. Nutritionally forced intra- and interspecific matings provide genetic information about the relatedness of homothallic isolates, the relation of the various species to one another, the genetic basis of homothallism, and its relationship to heterothallism. Most homothallic isolates are interfertile when nutritionally forced. Successful hybridization between species is restricted to particular combinations of homothallic x bipolar isolates. Significant findings of these studies include: (1) documentation of hybridization of biological species in the Homobasidiomycetes, (2) documentation of the relatedness of two naturally occurring, variant systems, homothallism and bipolar heterothallism, that regulate sexual morphogenesis in the higher fungi, (3) evidence for definite, but limited, evolutionary divergence of the polygenic, regulated components of the respective systems, and (4) indication that the genetic basis of homothallism in this system is essentially due to constitutive function and consequently is fundamentally different from presently understood mechanisms in other self-fertile systems.     

Factors affecting mushroom Pleurotus spp.

Pleurotus genus is one of most extensively studied white-rot fungi due to its exceptional ligninolytic properties. It is an edible mushroom and it also has several biological effects, as it contains important bioactive molecules. In basidiomycete fungi, lignocellulolytic enzymes are affected by many typical fermentation factors, such as medium composition, ratio of carbon to nitrogen, pH, temperature, air composition, etc. The survival and multiplication of mushrooms is related to a number of factors, which may act separately or have interactive effects among them. Out that understanding challenges in handling Pleurotus species mushroom requires a fundamental understanding of their physical, chemical, biological and enzymatic properties. This review presents a practical checklist of available intrinsic and extrinsic factors, providing useful synthetic information that may help different users. An in-depth understanding of the technical features is needed for an appropriate and efficient production of Pleurotus spp.     

CTC medium: A novel dodine-free selective medium for isolating entomopathogenic fungi,especially Metarhizium acridum,from soil

The selective media most commonly used for isolating hyphomycetous species of entomopathogenic fungi from non-sterile substrates rely on N-dodecylguanidine monoacetate (dodine) as the selective fungicide. Although these media are effective for isolating many species of Metarhizium and Beauveria from soil, they are inefficient media for isolation of an important Metarhizium species, Metarhizium acridum, from non-sterile soil. Our current study was directed to formulating a dodine-free selective medium that is efficient for isolating naturally occurring Beauveria spp. and Metarhizium spp., especially M. acridum, from soil. The selective medium (designated CTC medium) consists of potato dextrose agar plus yeast extract (PDAY) supplemented with chloramphenicol, thiabendazole and cycloheximide. In comparisons with selective media previously reported in the literature, the CTC medium afforded colonies that were larger and had both earlier and more abundant conidiation of entomopathogenic fungi, features which greatly facilitated identification of the emerging entomopathogenic fungi. In addition to efficient re-isolation of M. acridum, this medium also is an effective tool for selective isolation of Metarhizium brunneum, Metarhizium robertsii, Beauveria bassiana and Beauveria brongniartii from non-sterile field-collected soil samples inoculated (spiked) with fresh conidia in the laboratory.     

木材腐朽菌培养特性的研究综述

论述了研究木材腐朽菌培养特性的意义,对自1889年以来有关木材腐朽菌培养特性的研究文献进行了回顾。通过综合分析以往国内外对木材腐朽菌培养特性研究的结果,得出了对木材腐朽菌培养特性研究的总体结论。     

Ability of ectomycorrhizal fungi to utilize starch and related substrates

Basidiomycetous fungi of 55 strains of 33 species in 15 genera which are thought to be ectomycorrhizal were grown on starch and related substrates as a sole carbon source, and their ability to utilize these substrates was determined. Mycellial weights of the fungi grown on agar media containing starch and amylose varied between the strains from 1.1 to 94.9 mg/flask and from 0.4 to 93.3mg/flask, respectively. Mycelial growth rates ranged from 0 to 1.17 mm/d on barley grain medium and from 0 to 2.03 mm/d on rice grain medium; the highest rate corresponded to about half of the average of reference wood-rotting fungi. Most of the mycorrhizal fungi that grew well on amylose gave higher growth rates on barley. Several strains inLyophyllum, Hebeloma, Sarcodon, andTricholoma grew well on both glucose and starch media.     

Genetic Analysis in Wood-Decaying Fungi I. Genetic Variation and Evidence for Allopatric Speciation in Pleurotus ostreatus using Phenoloxidase Zymograms and Morphological Criteria

Using electropherograms (zymograms) of the phenoloxidase laccase and characteristics of mycelial growth and fruit body production, a distinct morphological and biochemical differentiation of two geographically isolated (allopatric) populations of the wood-rotting basidiomycete Pleurotus ostreatus became evident. No limitation in their outbreeding ability was observed, however. A specific secretory mechanism for an extracellular laccase, genetically different in the two geographical races, could be detected. An approximately 1: 1 segregation of this laccase band in the F₁ generation indicates that specific secretion of this enzyme is controlled by one gene only, Different degrees of genetic variation as shown by differences in the respective laccase spectra were found in the two geographical races. Only one enzyme band out of nine multiple laccases was found to be specific for fruit bodies. The value of zymograms for chemotaxonomic purposes, for the understanding of microevolution and for determination of genetic variation in fungi is critically discussed.     

Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5′ gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.     

Successful large-scale production of fruiting bodies of Laetiporus sulphureus (Bull.: Fr.) Murrill on an artificial substrate

Laetiporus sulphureus is an edible wood-rotting basidiomycete fungus whose fruiting bodies contain substances with verified therapeutic evidences and large amounts of α-(1 → 3)-glucan which is used as an effective inducer of microbial α-(1 → 3)-glucanases. However, production of mature fruiting bodies of this species under artificially controlled conditions has not been reported until now. Here, we provide the first report of successful initiation and development of L. sulphureus fruiting bodies in large-scale experiments. Twelve Laetiporus strains were isolated from a natural habitat. A synthetic log production system with a substrate composed of a mixture of sawdust enriched with organic and inorganic additives was developed. It was found that shocking the fungus mycelium with cold water or low temperature was the only suitable method for forced fruiting of L. sulphureus strains. Primordia of two strains were initiated already after 5–6 days from induction, and after another 2 days, they began to develop into fruiting bodies. Carpophores appeared fastest on substrates with high organic supplementation (40–45 %) and a low moisture content (40 %). The resulting mature fruiting bodies reached a weight of 200–300 g. The method of cultivation presented in this paper opens the way to commercial production of this valuable basidiomycete.