Changes in oxidative enzyme activity during interspecific mycelial interactions involving the white-rot fungus Trametes versicolor

Interspecific fungal antagonism leads to biochemical changes in competing mycelia, including up-regulation of oxidative enzymes. Laccase, manganese peroxidase (MnP), manganese-repressed peroxidase (MRP) and lignin peroxidase (LiP) gene expression and enzyme activity were compared during agar interactions between Trametes versicolor and five other wood decay fungi resulting in a range of interaction outcomes from deadlock to replacement of one fungus by another. Increased laccase and Mn-oxidising activities were detected at all interaction zones, but there were few changes in activity in regions away from the interaction zone in T. versicolor mycelia compared to self-pairings. Whilst no LiP activity was detected in any pairing, low level LiP gene expression was detected. MnP activity was detected but not expression of MnP genes; instead, MRP could explain the observed activity. No relationship was found between extent of enzyme activity increase and interaction outcome. Similarities between patterns of gene expression and enzyme activity are discussed.     

Laccase production by free and immobilized mycelia of Peniophora cinerea and Trametes versicolor: a comparative study

The production of laccase by immobilized mycelia of Peniophora cinerea and Trametes versicolor was studied. In an initial stage, experimental assays were performed in Erlenmeyer flasks using free and immobilized mycelium, and the performance of the fungal strains to produce the enzyme was compared. Both fungi adhered into the support material (a synthetic fiber), growing not only on the surface but also in the interspaces of the fibers. Immobilization of P. cinerea provided a 35-fold increase in laccase production when compared to the production obtained by using free mycelium. On the other hand, immobilization of T. versicolor caused a decrease in laccase activity. A comparison between the strains revealed that immobilized P. cinerea (3,500 U/L) surpassed the enzyme production by free T. versicolor (800 U/L). When the conditions that gave the best laccase production to each fungus were employed in a stirred tank bioreactor, very low laccase production was observed for both the cases, suggesting that shear stress and mycelia damage caused by the agitation impellers negatively affected the enzyme production.     

Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood

Trametes versicolor was shown to produce extracellular laccase during surface cultivation on glucose, wheat straw and beech wood. Growth on both wheat straw and beech wood led to an increase as high as 3.5-fold in extracellular laccase activity, in comparison with growth on glucose. The corresponding yields in fungal biomass reached only about 20% of the value obtained on glucose. Manganese peroxidase activity␣appeared during growth on wheat straw and beech wood. Mycelia grown on glucose, wheat straw and beech wood also showed intracellular laccase activities, monitored with 2,6-dimethoxyphenol, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 4-hydroxy-3,5-dimethoxybenzaldehyde azine (syringaldazine) and 3,4-dihydroxyphenylalanine (l-DOPA). Assaying intracellular laccase with 2,6-dimethoxyphenol, syringaldazine and l-DOPA showed the maximum oxidation rates to be at pH values different from those producing maximum oxidation rates with extracellular laccase. In each case most of the total laccase activity was recovered from the culture filtrates. Growth on wheat straw and beech wood led to increased values for both extra- and intracellular laccase activities, based on fungal dry weight, in comparison with growth on glucose. Received: 18 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996     

Activities of chitinolytic enzymes during primary and secondary colonization of wood by basidiomycetous fungi

The nitrogen (N) content of wood is usually suboptimal for fungal colonization. During decomposition of wood, an increasing fraction of the N becomes incorporated into fungal mycelium. Between 5 and 50% of the N in wood-degrading mycelium may be incorporated into chitin. Chitinolytic enzymes render this N available for re-utilization. Here, the activities of chitinolytic enzymes produced by wood-rotting fungi during degradation of spruce (Picea abies) wood were quantified in situ using fluorogenic 4-methylumbelliferyl substrates. A new method was developed that enables spatial quantification of enzyme activities on solid surfaces. All of the three tested fungi produced endochitinases, chitobiosidases and N-acetylhexosaminidases during colonization of wood. N-acetylhexosaminidase activity, and in some cases also chitobiosidase and endochitinase activities, were higher during secondary overgrowth of another fungus than during primary colonization of noncolonized wood. The results suggest that wood-degrading fungi degrade their own cell walls as well as the hyphae of earlier colonizers. Recycling of cell wall material within single mycelia and between fungal individuals during succession may lead to retention of N within woody debris.     

Antagonistic fungal interactions influence carbon dioxide evolution from decomposing wood

Fungal species vary in the rate and way in which they decay wood. Thus, understanding fungal community dynamics within dead wood is crucial to understanding decomposition and carbon cycling. Mycelia compete for wood territory, by employing antagonistic mechanisms involving changes in morphology, and production of volatile and diffusible chemicals. This is metabolically costly, and may affect the rate of use of the resource. The metabolic rate during pairwise interactions between wood decay ascomycetes and basidiomycetes was determined by measuring CO₂ production. CO₂ evolution altered over time, but changes were combination-specific. In only two combinations – when the dominant competitor overgrew the opponent's territory as mycelia cords – did CO₂ evolution increase over the course of the whole interaction. In most interactions, CO₂ evolution increased only after complete replacement of one competitor, suggesting utilisation of the predecessor mycelium or differences in decay ability due to alteration of the resource by the predecessor. There was no relationship between rate of CO₂ evolution and combative ability nor outcome of interaction.     

Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes?

The aim of this work was to investigate the poorly understood effects of co-culturing of two white rot fungi on the production of lignin-degrading enzyme activities. Four species, Ceriporiopsis subvermispora, Physisporinus rivulosus, Phanerochaete chrysosporium and Pleurotus ostreatus were cultured in pairs to study the degradation of aspen wood and the production of lignin-degrading enzymes. Potential of co-culturing for biopulping was evaluated. Chemical analysis of decayed aspen wood blocks showed that co-culturing of C. subvermispora with P. ostreatus could significantly stimulate wood decay, when compared to monocultures. Based on the fungi tested here, however, this effect is species-specific. Other combinations of fungi were slightly stimulating or not stimulatory. The pattern of lignin degradation was altered towards the acid insoluble part of lignin especially in co-cultures where P. ostreatus was included as a partner. The use of agar plates containing the polymeric dye Poly R-478 showed elevated dye decolourization at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. ostreatus with C. subvermispora. Manganese peroxidase activity was stimulated in co-cultures of P. ostreatus with C. subvermispora or with P. rivulosus. Immunoblotting indicated changes in lignin-degrading enzymes and/or their isoform composition in response to co-culturing. This is the first report on the effects of co-culturing of potential biopulping fungi on wood degradation, and gives basic knowledge on fungal interactions during wood decay that can be utilized in practical applications.     

Heterogeneous production of laccase by mycelium of white-rot fungi

Mycelium of white-rot fungi secretes laccase into the medium. It was found by cultivation on malt-agar plates that the mycelium does not produce laccase equally in all its parts. The youngest hyphae at the margins of the colony represent usually the maximum producers, whereas older hyphae produce less or none at all. An exception here isCollybia velutipes which is the weakest producer of laccase of all the fungi studied and where only the older hyphae begin to secrete it. Manometric estimation of laccase showed that maximum specific activity of laccase is achieved at the boundary between the phases of initial and linear growth and i₁₁ some cases during the first half of linear growth. Ageing of the mycelium characterized by certain changes in its metabolism is reflected in changes of enzyme production by fungal hypha of different age.     

Lignocellulose Decomposition and Production of Ligninolytic Enzymes During Interaction of White Rot Fungi with Soil Microorganisms

Abstract Four strains of white rot fungi, including two strains of Pleurotus sp., one Dichomitus squalens, and one Ganoderma applanatum, were grown on milled straw. After colonization of the straw by the fungi, sterile or nonsterile plugs of soil were added to the fungal substrates. The influence of the sterile soil and the indigenous soil microbiota on fungal growth, overall respiration, and production of ligninolytic exoenzymes was assessed. A method for extraction of laccase from soil samples was developed. Lignocellulose decomposition, and enzyme production of D. squalens were enhanced by the presence of sterile soil. The availability of inorganic compounds such as manganese may be a trigger for this stimulation. Neither growth nor the production of laccase and manganese peroxidase (MnP) of the Pleurotus strains was markedly affected by the soil microbiota. These fungi were highly competitive with the soil microbiota. It was demonstrated for the first time that the exoenzymes of such fungi are active in nonsterile soil. Enzyme activity in the aqueous phase of soil was high as in the aqueous phase of the straw substrate. D. squalens and G. applanatum did not withstand the competition with the soil microbiota, but the mycelia associated with straw were overgrown by soil microorganisms. Correspondingly, the fungi did not penetrate the soil, decomposition of lignocellulose was impeded, and the activities of laccase and MnP decreased dramatically. Received: 2 April 1996; Accepted: 7 June 1996     

Ligninolytic characteristics of Pleurotus ostreatus strain F6 and its monokaryotic protoplast derivative P19

A stable isolate of Pleurotus ostreatus P19 differing in some morphological and physiological characteristics from its parental wild-type strain F6 was obtained via protoplast isolation during the preparation of strains with altered ligninolytic abilities. The isolate is monokaryotic, does not form clamp-connections, and produces much higher activities of enzymes involved in lignin modification (laccase, manganese peroxidase). Cellulase activity was comparable to that of wild-type strain F6, but the xylanase activity was slightly higher in isolate P19. However, this monokaryotic derivative degrades lignin at a slightly lower rate than its parental strain F6. Electron microscopy observations of wood degradation as a function of mycelium growth were performed on three zones of birch wafers delimited according to the distance from the point of inoculation. The different stages of fungal mycelium growth showed differences in the ultrastructural patterns of the decay not only between the strains P19 and F6, but also depending on the distance from the point of inoculation. This suggests a spatio-temporally controlled secretion of enzymes along the hyphae. The enhanced ability of P19 to degrade the condensed forms of lignin in middle lamellae is correlated to its higher laccase activity.     

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

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

Characterisation of some Phlebiopsis gigantea isolates with respect to enzymatic activity and decay of Norway spruce wood

The activity of cellulase, peroxidase, phosphatase and dehydrogenase enzymes, together with the content of protocatechuic and vanillic acids, in samples of Norway spruce wood inoculated with 17 different isolates of Phlebiopsis gigantea was measured. The same isolates were used to compare decay activity in samples of Norway spruce wood after incubation for 3 and 6 months. Significant differences in enzyme activity and phenol production were found between aerial mycelium overgrowing the wood sample and the underlying wood. These differences indicated that the nature of the fungal mycelium appears to change depending on whether it is in contact with wood. After 6 months, highly extensive decomposition of the wood was shown by two British isolates. The results confirm a large difference in P. gigantea inoculum among isolates in natural conditions and reinforce the need for constant evaluation of the most active isolates to use in preparations for biocontrol: a problem for both users and registration bodies.     

Simulated nitrogen deposition affects wood decomposition by cord-forming fungi

Anthropogenic nitrogen (N) deposition affects many natural processes, including forest litter decomposition. Saprotrophic fungi are the only organisms capable of completely decomposing lignocellulosic (woody) litter in temperate ecosystems, and therefore the responses of fungi to N deposition are critical in understanding the effects of global change on the forest carbon cycle. Plant litter decomposition under elevated N has been intensively studied, with varying results. The complexity of forest floor biota and variability in litter quality have obscured N-elevation effects on decomposers. Field experiments often utilize standardized substrates and N-levels, but few studies have controlled the decay organisms. Decomposition of beech (Fagus sylvatica) blocks inoculated with two cord-forming basidiomycete fungi, Hypholoma fasciculare and Phanerochaete velutina, was compared experimentally under realistic levels of simulated N deposition at Wytham Wood, Oxfordshire, UK. Mass loss was greater with P. velutina than with H. fasciculare, and with N treatment than in the control. Decomposition was accompanied by growth of the fungal mycelium and increasing N concentration in the remaining wood. We attribute the N effect on wood decay to the response of cord-forming wood decay fungi to N availability. Previous studies demonstrated the capacity of these fungi to scavenge and import N to decaying wood via a translocating network of mycelium. This study shows that small increases in N availability can increase wood decomposition by these organisms. Dead wood is an important carbon store and habitat. The responses of wood decomposers to anthropogenic N deposition should be considered in models of forest carbon dynamics.     

Defining individual size in the model filamentous fungus Neurospora crassa

It is challenging to apply the tenets of individuality to filamentous fungi: a fungal mycelium can contain millions of genetically diverse but totipotent nuclei, each capable of founding new mycelia. Moreover, a single mycelium can potentially stretch over kilometres, and it is unlikely that its distant parts share resources or have the same fitness. Here, we directly measure how a single mycelium of the model ascomycete Neurospora crassa is patterned into reproductive units (RUs), meaning subpopulations of nuclei that propagate together as spores, and function as reproductive individuals. The density of RUs is sensitive to the geometry of growth; we detected 50-fold smaller RUs when mycelia had expanding frontiers than when they were constrained to grow in one direction only. RUs fragmented further when the mycelial network was perturbed. In mycelia with expanding frontiers, RU composition was strongly influenced by the distribution of genotypes early in development. Our results provide a concept of fungal individuality that is directly connected to reproductive potential, and therefore to theories of how fungal individuals adapt and evolve over time. Our data show that the size of reproductive individuals is a dynamic and environment-dependent property, even within apparently totally connected fungal mycelia.     

Identification and characterization of the biotechnological potential of a wild strain of Paraconiothyrium sp

The isolation and characterization of fungal strains from poorly described taxa allows undercover attributes of their basic biology useful for biotechnology. Here, a wild fungal strain (CMU‐196) from recently described Paraconiothyrium genus was analyzed. CMU‐196 was identified as Paraconiothyrium brasiliense by phylogenetic analysis of the rDNA internal transcribed spacer region (ITS). CMU‐196 metabolized 57 out of 95 substrates of the Biolog FF microplates. Efficient assimilation of dextrins and glycogen indicates that CMU‐196 is a good producer of amylolytic enzymes. It showed a remarkably assimilation of α‐d ‐lactose, substrate described as inducer of cellulolytic activity but poorly assimilated by several fungi. Metabolically active mycelium of the strain decolorized broth supplemented with direct blue 71, Chicago sky blue and remazol brilliant blue R dyes. The former two dyes were also well removed from broth by mycelium inactivated by autoclaving. Both mycelia had low efficiency for removing fuchsin acid from broth and for decolorizing wastewater from the paper industry. CMU‐196 strain showed extracellular laccase activity when potato dextrose broth was supplemented with Cu⁺², reaching a maximum activity of 46.8 (±0.33) U L^?1. Studied strain antagonized phytopathogenic Colletotrichum spp. fungi and Phytophthora spp. oomycetes in vitro, but is less effective towards Fusarium spp. fungi. CMU‐196 antagonism includes overgrowing the mycelia of phytopathogens and growth inhibition, probably by hydrosoluble extracellular metabolites. The biotechnological potential of strain CMU‐196 here described warrants further studies to have a more detailed knowledge of the mechanisms associated with its metabolic versatility, capacity for environmental detoxification, extracellular laccase production, and antagonism against phytopathogens. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:846–857, 2018     

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.     

Translocation of 32P between interacting mycelia of a wood-decomposing fungus and ectomycorrhizal fungi in microcosm systems

Interactions between saprotrophic and ectomycorrhizal fungi have been largely ignored, although their mycelia often share the same microsites. The mycelial systems show general similarity to each other and, although the enzymatic potential of the saprotrophic fungi is generally considered to be higher, the importance of organic nutrient sources to ectomycorrhizal fungi is now widely accepted. In the experiments described here, nutritional interactions involving transfer of elements from one mycelium to the other have been monitored dynamically using radioactive tracers and a non-destructive electronic autoradiography system. Microcosms were used in which mycelial systems of the ectomycorrhizal fungi Suillus variegatus and Paxillus involutus , extending from Pinus sylvestris host plants, were confronted with mycelia of the saprotroph Hypholoma fasciculare extending from wood blocks. The fungi showed a clear morphological confrontation response. The mycorrhizal mycelium often formed dense patches over the Hypholoma mycelia. Up to 25% of the ³²P present in the Hypholoma mycelium was captured by the mycorrhizal fungi and translocated to the plant host within 30 d. The transfer of ³²P to the saprotroph from labelled mycorrhizal mycelium was one to two orders of magnitude lower. The significance of this transfer as a 'short cut' in nutrient cycling is discussed.     

Interactions between extraradical ectomycorrhizal mycelia, microbes associated with the mycelia and growth rate of Norway spruce (Picea abies) clones

Despite their ecological relevance, field studies of the extraradical mycelia of ectomycorrhizal (ECM) fungi are rare. Here we examined in situ interactions between ECM mycelia and host vigour. Ectomycorrhizal mycelia were harvested with in-growth mesh bags buried under Norway spruce (Picea abies) clones planted in 1994 in a randomized block design. Mycelial biomass was determined and fungal species were identified by denaturing gradient gel electrophoresis (DGGE) and sequencing of the internal transcribed spacer 1 (ITS1) region. Microbial community structure in the mycelium was investigated by phospholipid fatty acid (PLFA) profiling. Compared to slow-growing spruce clones, fast-growing clones tended to support denser mycelia where the relative proportions of Atheliaceae fungi and PLFAs indicative of Gram-positive bacteria were higher. Ascomycetes and PLFAs representative of Gram-negative bacteria were more common with slow-growing clones. In general, the ECM mycelial community was similar to the ECM root-tip community. Growth rate of the hosts, the ECM mycelial community and the microbes associated with the mycelium were related, suggesting multitrophic interactions between trees, fungi and bacteria.     

Fourier transform infrared microscopy and imaging: detection of fungi in wood

FTIR microscopy was used to detect and discriminate the two wood decaying fungi Trametes versicolor and Schizophyllum commune in experimentally infected beech wood blocks. The distribution of fungal mycelium in wood was locally resolved and semiquantitatively recorded using FTIR microscopy combined with a focal plane array detector and image analysis. Cluster analysis revealed major differences between FTIR spectra recorded from wood fibers and empty vessel lumina and spectra from mycelium of both fungal species, irrespective of whether the fungi were grown on the surface of wood or inside vessel lumina. Species-specific clustering of spectra of fungal mycelium grown on the wood surface and inside vessel lumina demonstrated the potential of FTIR microscopy to discriminate among fungal species decaying wood.     

Collembola foraging responses to interacting fungi

Abstract 1. Temperate woodland Collembola feed predominantly on plant detritus and fungi. They exhibit preferences for different fungi reflecting resource value in terms of Collembola growth and reproduction. 2. Studies of fungal grazing by Collembola have focused on non‐interacting fungi and have used one genetic isolate as representative of an entire fungal species. This study examines the effects of fungal interactions on Collembola behaviour, and elucidates differences arising from the use of genetically distinct fungal isolates. 3. Folsomia candida were added to microcosms in which paired combinations of the fungi, Hypholoma fasciculare (four isolates), Phallus impudicus, Phanerochetae velutina and Resinicium bicolor (two isolates), were interacting. Collembola movement and mortality was recorded for 26 days. A constant preference for one fungal mycelium over the other was shown by Collembola in 12 of the 36 interactions studied. Lowest Collembola mortality was normally recorded on the preferred mycelium. 4. In 11 of the remaining 24 interactions, Collembola preference switched from one mycelium to the other; the greatest number of dead Collembola was recorded on the final preference. 5. Collembola behaviour on different fungal isolates was broadly similar, although in one species’ combination a change in fungal isolate resulted in a total reversal of Collembola preference. The implications of these results for decomposer food webs are considered.     

Isolation of sterols from the marine fungus Corollospora lacera

Several marine fungi collected from the waters of Prince Edward Island, Canada, were screened for the presence of natural products exhibiting antibacterial activity. Both broths and mycelia of these fungi were studied using the bioassay-guided chromatographic separation. The 4 fractions from the extract of mycelia of Corollospora lacera exhibited weak antibacterial activity and were analyzed further. From these fractions, 2 sterols (5 alpha,8 alpha-epidioxyergosterol and 22E,24R-ergosta-7,22-diene-3beta,5 alpha,6 beta-triol) and a 3:1 mixture of linoleic and oleic acids were isolated. The presence of ergosterol was confirmed in dichloromethane extracts of mycelia of every fungus in this study and this sterol was isolated from the extract of mycelium of Corollospora lacera. Two other known compounds (5-hydroxymethylfuran-2-carbaldehyde and bis(2-ethylhexyl) phthalate), were isolated from the dichloromethane extract of mycelium of Monodictys pelagica. The phthalate was reported in the literature as a metabolite isolated from the fungi, but in our study it was proven to be an artifact of the culturing and (or) extraction procedures rather than a true fungal metabolite.