Wood-inhabiting ligninolytic basidiomycetes in soils: Ecology and constraints for applicability in bioremediation

Ligninolytic basidiomycetes associated with wood decay are a physiologically distinct group of saprotrophic fungi capable of decomposition of all major components of wood. Although wood is their natural substrate, several species can survive in soil if suitable substrates are available, the soil-colonizing ability of species in the genera Phanerochaete, Pleurotus and Trametes being comparable to that of the soil-inhabiting basidiomycetes utilizing plant litter. Wood-inhabiting ligninolytic basidiomycetes (WLB) in soil interact with soil microflora, and some species are very efficient competitors affecting both the soil microbial biomass and the composition of the indigenous microbial community. The extracellular enzymes utilized by saprotrophic basidiomycetes for nutrient acquisition participate in the interspecific interactions with other soil biota but are also involved in the transformation of soil organic matter – lignocellulose and humic compounds. Bioremediation research has significantly enriched our knowledge of the ecology of basidiomycetes, but it has also identified several limitations for practical applicability of WLB at a field scale, and has led to the conclusion that natural attenuation or bioaugmentation are todays methods of choice for on site treatment.     

Diversity of basidiomycetes in michigan agricultural soils

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With "species" defined as organisms yielding PCR products with > or =99% identity in the 5' 650 bases of the nuclear large-subunit ribosomal DNA, 241 "species" were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 "species" had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple "species" could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated "species" richness for this 11.2-ha site was 367 "species" of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure.     

The quantity and structure of the saprotrophic bacterial complex of the mycorhizosphere and hyphosphere of symbiotrophic basidiomycetes

The impact of symbiotrophic basidiomycetes on the abundance and structure of the soil saprotrophic bacterial complex of the mycorhizosphere and hyphosphere of 10 species of symbiotrophic basidiomycetes in natural conditions is studied. Compared to the reference soil, one can observe a significant increase in bacterial abundance in the mycirhizosphere and hyphosphere of basidiomycetes. The analysis of the taxonomical structure of bacterial complexes showed that Pseudomonas prevail in the mycorhyzosphere and Bacillus, in the rhizosphere and reference soil. Bacterial complexes of the hyphosphere and those in reference soil showed greater similarity to each other than either of them does to the mycorhizosphere. This fact implies the selective impact of the mycorhizosphere on soil bacteria.     

Saprotrophic basidiomycete mycelia and their interspecific interactions affect the spatial distribution of extracellular enzymes in soil

Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates in soil. The production of extracellular hydrolytic enzymes was studied during the growth of two saprotrophic basidiomycetes, Hypholoma fasciculare and Phanerochaete velutina, across the surface of nonsterile soil microcosms, along with the effects of these basidiomycetes on fungi and bacteria within the soil. Higher activities of α-glucosidase, β-glucosidase, cellobiohydrolase, β-xylosidase, phosphomonoesterase and phosphodiesterase, but not of arylsulphatase, were recorded beneath the mycelia. Despite the fact that H. fasciculare, with exploitative hyphal growth, produced much denser hyphal cover on the soil surface than P. velutina, with explorative growth, both fungi produced similar amounts of extracellular enzymes. In the areas where the mycelia of H. fasciculare and P. velutina interacted, the activities of N-acetylglucosaminidase, α-glucosidase and phosphomonoesterase, the enzymes potentially involved in hyphal cell wall damage, and the utilization of compounds released from damaged hyphae of interacting fungi, were particularly increased. No significant differences in fungal biomass were observed between basidiomycete-colonized and noncolonized soil, but bacterial biomass was reduced in soil with H. fasciculare. The increases in the activities of β-xylosidase, β-glucosidase, phosphomonoesterase and cellobiohydrolase with increasing fungal:bacterial biomass ratio indicate the positive effects of fungal enzymes on nutrient release and bacterial abundance, which is reflected in the positive correlation of bacterial and fungal biomass content.     

Diversity of Basidiomycetes in Michigan Agricultural Soils

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With “species” defined as organisms yielding PCR products with ≥99% identity in the 5′ 650 bases of the nuclear large-subunit ribosomal DNA, 241 “species” were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 “species” had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple “species” could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated “species” richness for this 11.2-ha site was 367 “species” of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure.     

Role of Plants in the Vegetative and Reproductive Growth of Saprobic Basidiomycetous Ground Fungi

Non-symbiotic microorganisms engineered or expensively selected to degrade xenobiotic hydrocarbons or modify heavy-metal uptake of plants in soil remediations die back after their introduction into the target soils. Mycelia of saprobic basidiomycetes were therefore inoculated into soil samples of 1 l in glass vessels to record mycelial growth and reproduction in the immediate rhizosphere of up to 11 herbaceous plant species, or to study their responses to the separate volatiles from whole plant swards or their root balls whose emanations had been collected in 1.5-l plastic bags fixed to the glass vessels. Excess CO₂ was controlled with NaOH solution. Volatiles from root balls of parsley and pea but not wheat, from unplanted soils, from the fungus-permeated, unplanted substrate soil itself, and from the rooting soil of whole wheat sward increased mycelial densities in Clitocybe sp. more than in Agaricus macrocarpus and indicated thus a higher nutrient state of the mycelia. Organic volatiles proved therefore to be a significant carbon source for certain basidiomycetes in poor natural soils. The contemporary decline in the number of basidiocarp initials to 0 to 36% in both fungi relative to the unplanted and aerated controls was caused by volatiles from rooted and unplanted soil and pointed thus to their ecological role as antibiotics, fumigants, toxins, and hormonal compounds. Aqueous extracts from root balls of wheat stimulated mycelial density and fruiting in A. macrocarpus contemporarily because of their contents in soil-derived macronutrients. They suppressed once more fruiting in the more sensitive Clitocybe sp. by active agents in the aqueous phase. Within plant rhizospheres, densities of Clitocybe sp. mycelia were stimulated in the presence of alfalfa, carrot, red clover, ryegrass, and spinach, whereas those of A. macrocarpus were halved by 7 of 10 plant species including alfalfa, red clover, ryegrass, and spinach. Mycelia of A. macrocarpus may thereby have responded to differences in concentration and composition of volatile compounds. The contemporary repression of fruiting in both fungi and in nearly all treatments was not due to plant competition for macronutrients. Mycelia of basidiomycetes over-compensated for losses in macronutrients to the plant by decomposing soil matrix constituents. It is concluded that organic volatiles emitted by several plant organs and natural soils improved the nutritional state of A. macrocarpus and Clitocybe sp. but not of Agaricus bisporus mycelia and could therefore help establish certain ground fungi in the field. The contemporary and general suppression of fruiting by constituents of the gaseous (and liquid) phase in all fungi examined suggests interference with basic physiological processes and recommends an urgent re-examination of the degradative ability of basidiomycetes in the presence of volatiles.     

Impacts of grazing soil fauna on decomposer fungi are species-specific and density-dependent

The grazing impacts of different densities of woodlice, collembola and millipedes on the foraging and distribution of two saprotrophic cord-forming basidiomycetes were investigated in soil microcosms. Effects of all three invertebrate species were density-dependent, with larger populations limiting mycelial development to a greater extent. Impacts were, however, species-specific; grazing pressures exerted by low-density woodlouse populations outweighed those of high-density millipede or collembola populations. The varying abilities of soil invertebrates to influence mycelial foraging and distribution indicate that invertebrate species composition and diversity may be key factors regulating saprotrophic basidiomycete functioning in woodland soil.     

Molecular analysis of fungal communities and laccase genes in decomposing litter reveals differences among forest types but no impact of nitrogen deposition

The fungal community of the forest floor was examined as the cause of previously reported increases in soil organic matter due to experimental N deposition in ecosystems producing predominantly high-lignin litter, and the opposite response in ecosystems producing low-lignin litter. The mechanism proposed to explain this phenomenon was that white-rot basidiomycetes are more important in the degradation of high-lignin litter than of low-lignin litter, and that their activity is suppressed by N deposition. We found that forest floor mass in the low-lignin sugar-maple dominated system decreased in October due to experimental N deposition, whereas forest floor mass of high-lignin oak-dominated ecosystems was unaffected by N deposition. Increased relative abundance of basidiomycetes in high-lignin forest floor was confirmed by denaturing gradient gel electrophoresis (DGGE) and sequencing. Abundance of basidiomycete laccase genes, encoding an enzyme used by white-rot basidiomycetes in the degradation of lignin, was 5-10 times greater in high-lignin forest floor than in low-lignin forest floor. While the differences between the fungal communities in different ecosystems were consistent with the proposed mechanism, no significant effects of N deposition were detected on DGGE profiles, laccase gene abundance, laccase length heterogeneity profiles, or phenol oxidase activity. Our observations indicate that the previously detected accumulation of soil organic matter in the high-lignin system may be driven by effects of N deposition on organisms in the mineral soil, rather than on organisms residing in the forest floor. However, studies of in situ gene expression and temporal and spatial variability within forest floor communities will be necessary to further relate the ecosystem dynamics of organic carbon to microbial communities and atmospheric N deposition.     

Laccase Gene Composition and Relative Abundance in Oak Forest Soil is not Affected by Short-Term Nitrogen Fertilization

Anthropogenic nitrogen (N) deposition affects a wide range of soil processes including phenol oxidase (PO) activity and soil organic matter dynamics. Depression of phenol oxidase activity in response to N saturation is believed to be mediated by the activity of white-rot basidiomycetes, whose production of extracellular oxidative enzymes can be limited by high N availability. We examined the effect of short-term N deposition on basidiomycete laccase gene diversity and relative abundance in temperate oak forest soil in which significant decreases in phenol oxidase and increased SOM have been recorded in response to experimental N deposition. UniFrac was used to compare the composition of laccase genes between three control- and three nitrogen-fertilized (80 kg⁻¹ ha⁻¹ per year) oak forest soils. The relative abundance of laccase genes was determined from qPCR analysis of laccase and basidiomycete ITS gene abundances. Our results indicate that there was no significant shift in the composition of laccase genes between control- and N-fertilized soils, nor was there a significant change in the relative abundance of laccase genes. These data suggest that N deposition effects on mineral soil PO activity do not result from changes in laccase gene diversity of white-rot basidiomycetes but are likely the result of altered microbial abundance or expression in this ecosystem type. Furthermore, laccase gene composition may be tied to factors that structure microbial communities in general, as soil laccase gene communities are more similar to other forest soils than with the corresponding litter.     

Enrichment of perforate septal pore caps from the basidiomycetous fungus Rhizoctonia solani by combined use of French press, isopycnic centrifugation, and Triton X-100

Septal pore caps occur in many filamentous basidiomycetes located at both sides of the dolipore septum and are at their base connected to the endoplasmic reticulum. The septal pore cap ultrastructure has been described extensively by the use of electron microscopy, but its composition and function are not yet known. To enable biochemical and functional analyses in the future, we here describe an enrichment method for perforate septal pore caps from Rhizoctonia solani. Our method is based on the combined use of French press and isopycnic centrifugation, using a discontinuous sucrose gradient followed by a treatment with Triton X-100. Enrichment was monitored by the use of scanning electron microscopy and transmission electron microscopy. Using the same isolation method, smaller septal pore caps were isolated from two other basidiomycetes as well. Furthermore, we showed pore-occluding material co-purified with the septal pore caps. This observation supports the hypothesis that septal pore caps play a key role in the plugging process of the septal pores in filamentous basidiomycetes.     

Studies on the Growth of Basidiomycetes in Soil

Fructifications of basidiomycetes have been collected at LakenheathWarren, and thirty-nine species have been obtained from fivegrassland soils. The different soils have been found to havedifferent species growing upon them and most species occur onthe alkaline-slightly acid grasslands. Highly acid grasslandat Lakenheath has a poor fungus flora. Studies have been made of the regions in the soil in which themycelia of certain of these basidiomycetes occur, and attemptshave been made to isolate these fungi from soil. Some specieshave been found to inhabit the litter zone rather than the minerallayers of the soil. A few form extensive rhizomorph systemswhich may penetrate deeply into the soil. A few species of ringfungi, such as Marasmius oreades, Psalliota arvensis, and Tricholomanudum, have been found to possess a well-defined mycelial zonein the soil, from which the fungus could be isolated. Profile studies have shown that the mycelial zones of thesespecies contain a restricted population of microfungi, bothfewer species and fewer colonies, as compared with the normalsoil around. Ascomycetes, such as Arachniotus, Chaetomium, Gymnoascus,and Penicillium, have been isolated more frequently from mycelialzones than from normal soil.     

Investigations on natural durability of important European wood species against wood decay fungi. Part 1: Laboratory tests

The durability of heartwood from European larch, Sessile oak and Scots pine was tested in laboratory against wood decay basidiomycetes and soft rot. The durability test was performed according to CEN/TS 15083-1:2005 with Coniophora puteana, Oligoporus (Poria) placenta and Trametes (Coriolus) versicolor. CEN/TS 15083-2:2005 was applied in order to test the timber with a bioactive soil substrate against soil rotting organisms. For Sessile oak, a very high durability against basidiomycetes (DC 1) was found in contrast to a low durability (DC 4) against soft rot fungi. Furthermore the results indicated that the durability of European larch and Scots pine is slightly deteriorated after leaching according to EN 84:1997. Beside this a minor influence of raw density on mass loss was detected for larch.     

Allergenicity of airborne basidiospores and ascospores: need for further studies

Many known fungal species are grouped among basidiomycetes and ascomycetes. Active mechanisms of spore release into air currents are among the main features of these fungi. Aerobiological studies have described their presence in many regions worldwide. In some areas, fungi have been described as the predominant outdoor airborne biological particulate with much higher concentrations than pollen. Other studies have determined that among the fungal aerospora, the highest concentrations belong to basidiospores and ascospores. Nevertheless, the allergenic potential of spores from basidiomycetes and meiotic forms of ascomycetes has not been studied to the extent of mitosporic fungi and allergens from other sources. The need to further evaluate the role of basidiomycetes and meiotic ascomycetes in allergies is evidenced by the few genera with characterized allergens and limited studies that had demonstrated levels of sensitization similar or higher to that of mitosporic fungi and other allergens. In this review, based on the existing aerobiological, epidemiological, immunological, and molecular biology studies, we provide evidence that the role of basidiomycetes and ascomycetes deserves more attention with respect to their roles as potential aeroallergens.     

Phylogenetic relationships of ascomycetes and basidiomycetes based on comparative genomics analysis

The relationship between ascomycetes and basidiomycetes, the two main phyla of non-flagellated fungi, has rarely been investigated. In this study, we performed a comparative genomics analysis of genome sequences of 55 ascomycetes and 26 basidiomycetes species and detected 81 universal markers, 875 homologous genes and a conserved contig in the glucose-regulated protein gene. In dendrograms based on simple sequence repeat markers and homologous genes, ascomycetes and basidiomycetes formed distinct clusters, with each set of taxa having a high coefficient of relatedness. Ascomycetes and basidiomycetes also constituted distinct groups in a phylogenetic tree based on a conserved contig in the glucose-regulated protein gene. These results provide evidence that basidiomycetes may be derived from ascomycetes but are definitely genetically differentiated at the genomic level. The phylogenetic relationships of ascomycetes and basidiomycetes uncovered in this study provide new insights for future research related to fungal classification and evolution.     

Grazing by Folsomia candida (Collembola) differentially affects mycelial morphology of the cord-forming basidiomycetes Hypholoma fasciculare, Phanerochaete velutina and Resinicium bicolor

Cord-forming basidiomycetes are important decomposers of dead wood in forest ecosystems but the impact of mycophagous soil invertebrates on their mycelia are little known. Here we investigate the effects of different grazing intensities of Collembola (Folsomia candida) on mycelial foraging patterns of the saprotrophic cord-forming basidiomycetes Hypholoma fasciculare, Phanerochaete velutina and Resinicium bicolor growing from beech (Fagus sylvatica) wood block inocula in dishes of non-sterile soil. Mycelial extension rate and hyphal coverage decreased with increased grazing intensity. R. bicolor was most affected, high grazing density resulting in only a few major cords remaining. Grazing of H. fasciculare often resulted in points of more rapid outgrowth as cords with a fanned margin. In grazed mycelia of P. velutina the main cords had fanned tips and lateral cords became branched. These results suggest that mycophagy by Collembola may hinder the growth of cord-forming fungi in woodlands, which might impact on the ability of these fungi to forage for and decompose dead organic material.     

Distribution patterns of Dikarya in arid and semiarid soils of Baja California,Mexico

Approximately four-fifths of the land area of Baja California (BC) in Mexico are occupied by arid and semiarid soils, the mycobiota of which is virtually uncharacterized. In the first culture-independent study of the mycobiota of BC, we collected soil from five different locations in the region and constructed a Dikarya-specific gene library for the ITS region of nuclear ribosomal DNA. Clones were analyzed by RFLP, were sequenced for phylogenetic analyses, and diversity and similarity indices were calculated. The ascomycete Penicillium dipodomyicola was the most frequent fungus found in soil at the most arid location studied, and the basidiomycete Coprinellus radians was the most frequent at the location receiving the highest rainfall. Other frequent members of the soil mycobiota were identified as Alternaria spp., Ceratobasidium sp., Coniozyma leucospermi, Nematoctonus robustus, Penicillium griseofulvum, Tulostoma kotlabae and uncultured members of the Dikarya. Several sequences were identified as those of uncultured fungi, one of which was previously reported from other hot deserts. Arid soils and the transitional zones between arid and semiarid soils had the most similar fungal diversity, with the former soils having a community from which basidiomycetes were absent, and the soil receiving the highest precipitation having a community dominated by basidiomycetes.     

一株源于南极土壤的耐冷真菌Sistotrema brinkmannii(英文)

从南极Dry Valleys Feyxell Basin湖土壤中分离获得一株具有锁状联合的耐冷担子菌,核糖体DNAITS区序列分析将该菌株鉴定为Sistotrema brinkmannii。形态特征研究表明该菌株产生裸担子,其上生4-8个小梗,每个梗上产生一个肾形担孢子,丝状担子菌在南极少有报道,该菌株的生物学及生态学值得进一步研究。     

Three-dimensional outgrowth of a wood-rotting fungus added to a contaminated soil from a former gasworks site

The capability of wood-rotting fungi (WRF) to colonise contaminated soil is an important fungal characteristic in the development of WRF-based soil bioremediation, it is also important to have methods that monitor the presence of the WRF in the soil. In this lab-scale study, it was shown that it was possible to re-capture, localise and identify a brown-rot fungus, Antrodia vaillantii, after it has been inoculated into, and grown in, a contaminated soil from a former gasworks site. The three-dimensional outgrowth of A. vaillantii was monitored by allowing it to grow into fungicide-treated wood baits, temporarily placed in the soil. After two weeks, the baits were withdrawn from the soil and surface sterilised with hydrogen peroxide to favour fungi growing inside baits, i.e., A. vaillantii. After subsequent plating of baits on selective agar medium the presence of A. vaillantii was confirmed with PCR/RFLP. A. vaillantii was found to be viable throughout the 54 days long study and exhibited a surface growth pattern similar to other well-known cord-forming basidiomycetes. Firstly, the upper part of the soil closest to the place of inoculation was colonised, however, over a period of time, the area of colonisation spread deeper into the soil. The detection method employed in the current study gave a conservative estimate of the fungal proliferation and did not require extensive sampling. Its use could be applicable in both applied research, such as soil bioremediation, and in pure microbial ecology studies.     

Preservation of live cultures of basidiomycetes – Recent methods

Basidiomycetes are used in industrial processes, in basic or applied research, teaching, systematic and biodiversity studies. Efficient work with basidiomycete cultures requires their reliable source, which is ensured by their safe long-term storage. Repeated subculturing, frequently used for the preservation, is time-consuming, prone to contamination, and does not prevent genetic and physiological changes during long-term maintenance. Various storage methods have been developed in order to eliminate these disadvantages. Besides lyophilization (unsuitable for the majority of basidiomycetes), cryopreservation at low temperatures seems to be a very efficient way to attain this goal. Besides survival, another requirement for successful maintenance of fungal strains is the ability to preserve their features unchanged. An ideal method has not been created so far. Therefore it is highly desirable to develop new or improve the current preservation methods, combining advantages and eliminate disadvantages of individual techniques. Many reviews on preservation of microorganisms including basidiomycetes have been published, but the progress in the field requires an update. Although herbaria specimens of fungi (and of basidiomycetes in particular) are very important for taxonomic and especially typological studies, this review is limited to live fungal cultures.     

Bacterial complexes of the fruiting bodies and hyphosphere of certain basidiomycetes

The surface and internal tissues of the fruiting bodies of basidiomycetes were shown to be specific bacterial habitats characterized by varying diversity and structure of bacterial complexes. On the surface of fruiting bodies, Gram-negative bacteria of the genera Pseudomonas, Xanthomonas, and Myxococcus prevailed, while in the internal tissues Gram-positive bacteria of the genera Streptomyces, Bacillus, Arthrobacter, and Micrococcus were identified in addition. Bacterial complexes from the surface and inner tissues of the fruiting bodies of the studied basidiomycetes showed significant similarity to each other and differed from those from the hyphosphere and the reference soil. On the surface and in the internal tissues of the fruiting bodies, representatives of the genus Myxococcus were identified for the first time, which could indicate initial decay of the fruiting body.