Soil Basidiomycetes Research Articles

Isolation and molecular identification of laccase-producing saprophytic/phytopathogenic mushroom-forming fungi from various ecosystems in Michoacán State, Mexico

The aim of this study was isolation and molecular identification of laccase-producing saprophytic/phytopathogen Basidiomycetes species from different geographic regions with dominant vegetation of <em>Pinus</em>, <em>Abies</em>, and <em>Quercus</em> spp. in the state of Michoacán, Mexico. Soil samples and visible mycelial aggregates were collected for fungal isolations. Soil samples were processed using a soil particle washing technique, where a selective Ascomycetes inhibitor and guaiacol, as an indicator of saprophytic Basidiomycetes growth, were used. Most of the isolates were obtained from samples collected in Parque Nacional, José Ma. Morelos (Km 23), Charo, Michoacán, Mexico. Based on sequence comparisons and phylogenetic analysis of internal transcribed spacer regions (ITS1-5.8S-ITS4) with respect to reference taxa, identification of saprophytic/phytopathogen Basidiomycetes species was carried out. In total, 15 isolates from 12 genera (i.e., <em>Bjerkandera</em>, <em>Coriolopsis</em>, <em>Ganoderma</em>, <em>Hexagonia</em>, <em>Irpex</em>, <em>Limonomyces</em>, <em>Psathyrella</em>, <em>Peniophora</em>, <em>Phlebia</em>, <em>Phlebiopsis</em>, <em>Trametes</em>, and <em>Trichaptum</em>) and one species from family Corticiaceae were identified. This study will be useful for further investigations on biodiversity of soil Basidiomycetes in different ecosystems. At present, these isolates are being used in our various lab experiments and can be useful in different industrial and bioremediation applications.

Nanoparticles synthesis by Agaricus soil basidiomycetes

We examined the effect of various concentrations of HAuCl4, AgNO3, Na2SeO3, Na2SiO3, and GeO2 on mycelial growth of the soil basidiomycetes Agaricus bisporus and A.arvensis in submerged and solid media. Fungal mycelial extracts and cell-free culture filtrates were able to reduce ions of Au, Ag, Se, Si, and Ge compounds, forming Au0, Ag0, Se0, Si0/SiO2 and Ge0/GeO2 nanoparticles. The physical characteristics of the mycogenic nanoparticles differed depending on the species of Agaricus and the type of extract. Au nanospheres obtained with cell-free culture filtrates were of 2-5nm diameter in A.bisporus and of 2-10nm in A.arvensis. Nanoparticles produced by extracts of mycelia were several times larger and highly heterogenous. Ag nanoparticles produced by cell-free culture filtrates were spherical or irregular-shaped and agglomerated, whereas with extracts of mycelia, small homogenous nanospheres of 1-10nm were formed. Se nanospheres obtained with cell-free culture filtrates were of 100-250nm diameter in A.bisporus and of 150-550nm diameter in A.arvensis. The particles synthesized with extracts of mycelia were of 40-140nm in A.bisporus and of 100-250nm in A.arvensis. Incubation of Na2SiO3 with cell-free culture filtrates resulted in porous Si nanoparticles of 30-65nm in A.bisporus and of 50-200nm in A.arvensis. Ge nanoparticles synthesized by both Agaricus species were mostly spheres of 50-250nm diameter.

Oil degradation by basidiomycetes in soil and peat at low temperatures

A total of 17 basidiomycete strains causing white rot and growing on oil-contaminated substrates have been screened. Three strains with high (Steccherinum murashkinskyi), average (Trametes maxima), and low (Pleurotus ostreatus) capacities for the colonization of oil-contaminated substrates have been selected. The potential for degrading crude oil hydrocarbons has been assessed with the use of fungi grown on nonsterile soil and peat at low temperatures. Candida sp. and Rhodococcus sp. commercial strains have been used as reference organisms with oil-degrading ability. All microorganisms introduced in oil-contaminated soil have proved to be ineffective, whereas the inoculation of peat with basidiomycetes and oil-degrading microorganisms accelerated the destruction of oil hydrocarbons. The greatest degradation potential of oil-aliphatic hydrocarbons has been found in S. murashlinskyi. T. maxima turned out to be the most successful in degrading aromatic hydrocarbons. It has been suggested that aboriginal microflora contributes importantly to the effectiveness of oil-destructing microorganisms. T. maxima and S. murashkinskyi strains are promising for further study as oil-oxidizing agents during bioremediation of oil-contaminated peat soil under conditions of low temperatures.

Bacterial communities in the fruit bodies of ground basidiomycetes

Fruit bodies of basidiomycetes at different stages of decomposition serve as specific habitats in forest biocenoses for bacteria and differ significantly with respect to the total bacterial population and abundance of particular bacterial genera. A significant increase in the total bacterial population estimated by the direct microscopic method with acridine orange staining and in the population of saprotrophic bacteria (inoculation of glucose peptone yeast agar) in fruit bodies of basidiomycetes Armillaria mellea and Coprinus comatus was recorded at the final stage of their decomposition in comparison with the initial stage. Gramnegative bacteria predominated in the tissues of fruit bodies at all the stages of decomposition and were represented at the final stage by the Aeromonas, Vibrio, and Pseudomonas genera (for fruit bodies of A. mellea) the Pseudomonas genus (for fruit bodies of C. comatus). The potential influence of bacterial communities in the fruit bodies of soil basidiomycetes on the formation of bacterial communities in the upper soil horizons in forest biocenoses is discussed. The loci connected with the development and decomposition of fruit bodies of basidiomycetes on the soil surface are promising for targeted search of Gram-negative bacteria, the important objects of biotechnology.

Basidioascus undulatus: genome, origins, and sexuality.

Basidioascus undulatus is a soil basidiomycete belonging to the order Geminibasidiales. The taxonomic status of the order was unclear as originally it was only tentatively classified in the class Wallemiomycetes. The fungi in Geminibasidiales have an ambiguously defined sexual cycle. In this study, we sequenced the genome of B. undulatus to gain insights into its sexuality and evolutionary origins. The assembled genome draft was approximately 32 Mb in size, had a median nucleotide coverage of 24X, and contained 6123 predicted genes. Previous morphological descriptions of B. undulatus relied on interpretation of putative sexual structures. In this study, nuclear staining and confocal microscopy showed meiosis occurring in basidia and genome analysis confirmed the existence of genes involved in meiosis and mating. Using 35 protein-coding genes extracted from genomic information, phylogenomic and molecular dating analyses confirmed that B. undulatus indeed belongs to a lineage distantly related to Wallemia while retaining a basal position in Agaricomycotina. These results, combined with differences in septal pore morphology, led us to move the order Geminibasidiales out of the Wallemiomycetes and into the new class Geminibasidiomycetes cl. nov. Finally, the concept of Agaricomycotina is emended to include both Wallemiomycetes and Geminibasidiomycetes.

Improved analysis of fungal communities using the next-generation-sequencing analysis of rpb2 genes

http://www.biomed.cas.cz/mbu/lbwrfCurrent exploration of the ecology of soil fungal and bacterial communities and microbe-catalyzed processes in soils largely rely on community composition analysis using next-generation-sequencing of PCR amplicons (1). Typically, the relative abundance of individual members of microbial communities are derived from the analyses of 16S rRNA region of prokaryotic microorganisms and 18S rRNA or internal transcribed spacer (ITS) region of the rDNA for fungi and other microeukaryots. The analysis of fungal ITS sequences is helpful tool for molecular systematics at the species level, and even within species, but the quantitative information on the relative abundance of individual taxa is skewed due to the presence of multiple rDNA gene copies per genome, ranging from 10 to 200 (2). On the other hand, it was demonstrated that there is a group of genes like the elongation factor-1 alpha (tef1) or RNA polymerase II second largest subunit (rpb2) that are consistently present in one copy per fungal genome and exhibit sufficient variation to be used for phylogenetic analysis and taxonomic assignment (3). The use of such genes offers the possibility to directly count fungal genomes and improve the knowledge on the relative importance of individual taxa of fungi in the environmental processes. Here we demonstrate that the amount of ITS copies per nanogram DNA shows high variation among soil basidiomycetes and even closely related species largely differ in this respect. We also demonstrate that the use of the rpb2 gene is applicable for analysis of soil fungal communities and that the data derived using this molecular marker are largely different from those based on the amplicon sequencing of the ITS. Although the phylogeneti discriminative power of the rpb2 gene is limited, it still offers a suitable tool to infer fungal taxonomy at least on the level of families.

Local distribution of ectomycorrhizae-associated basidiomycetes in forest soil correlates with the degree of soil organic matter humification and available electrolytes

Spatial distribution of ectomycorrhizae-associated basidiomycetes was determined in oakbirch forest using terminal restriction fragment length polymorphism (T-RFLP) analysis. The data were correlated with actual soil humidity, pH, electric conductivity of the soil extract, absorbance A(465) and A(665) of water and alkali soil extracts and with the ratio A(465)/A(665) (parameter A4/A6). Natural non-homogeneity of the soil parameters was used as experimental gradient. Distance-based redundancy analysis of the T-RFLP data (with soil parameters being taken as environmental parameters) provided significant results when ITS1F-terminanted restriction fragments were analyzed. Among other fungi, a Mycena galericulata related fungus was observed to correlate negatively with A4/A6, indicating its association with highly humified soil organic matter. Positive association of other, unidentified fungi with A4/A6 was also observed. Several other unidentified fungi negatively correlated with electric conductivity of the soil extract. The results may explain nonhomogeneity of the spatial distribution of the fungi associated with ectomycorrhizae as a result of their interaction with non-homogeneous soil environment.

Mutual influence of soil basidiomycetes and white mustard plants on their enzymatic and catabolic activities

Liquid and volatile emanations in interactions of soil basidiomycetes with herbs affect fungal oxidoreductases and stress-related plant peroxidases (PO). In this study, gnotobiotic co-cultures between 6 non-pathogenic saprobes and 2 ectomycorrhizal basidiomycetes with the non-host plant white mustard were established on glucose-salt medium with the respective controls. Determined were oxidoreductase activities for culture fluids and plant tissues at initial fungal idiophase and degradation rates of Remazol-BBR and 5 PAHs. In culture fluids of Agaricus arvensis, A. porphyrizon, Lepista nebularis, Stropharia rugoso-annulata, and Hypholoma fasciculare (group-5), the laccase-deficient plant enabled activity increases in fungal laccase (by 2300-fold), in extracellular (fungal and?) plant-derived peroxidases (by 21-fold), and in the dissipation of phenanthrene and anthracene. Oxidative activities in roots rose by 46000-fold during adsorption of fungal laccases. Increases in the stress-related shoot-PO (by 4.1-fold) were exclusively elicited by group-5 saprobes and correlated with plant-phenolic-mediated formations of Mn(III) and increases in Remazol BBR bleaching. Agaricus bisporus and the ectomycorrhizal Hebeloma crustuliniforme and Suillus granulatus did not respond to plant emanations with elevated laccase activities but solubilized apparently root-surface PO. They failed to elicit stress-related activity increases of PO in white mustard shoot and prevented Mn(III) formation in several tissues. It is concluded that white mustard emanations promoted the catabolic performance of the plant-stress eliciting group-5 saprobes but not of A. bisporus and the ectomycorrhizal fungi with their low stress-inducing potential. The nature of the plant-released stimuli and the classes of fungus-released stress agents discussed must be determined in further studies.

Dwarf mistletoe effects on soil basidiomycete community structure, soil fungal functional diversity, and soil enzyme function: Implications for climate change

We used a combination of molecular, culture and biochemical methods to test the hypothesis that severe infection of pine by dwarf mistletoe (genus Arceuthobium ) has significant effects on structure and function of soil fungal communities, and on carbon cycling in soils. PCR and DNA sequencing of the basidiomycete communities in paired blocks of uninfected and infected trees revealed: (1) that the top, organic soil layer in this system is inhabited almost exclusively by ectomycorrhizal fungi; (2) no difference in species richness (6 species core −1 in both) or Shannon–Wiener evenness (0.740 and 0.747 in uninfected and infected blocks respectively), however Shannon–Wiener diversity was significantly greater in infected blocks (1.19 vs 1.94 in uninfected and infected blocks respectively, P < 0.05); (3) significant differences in basidiomycete species composition, with nearly complete absence of two system co-dominant Russula species in infected blocks, and replacement of one co-dominant Piloderma species with another in infected plots, indicating physiological variability within the genus. Soil fungal physiological diversity measured using the Fungilog system was significantly greater in terms of both number of carbon substrates used by culturable soil fungi (both ascomycetes and basidiomycetes) in infected blocks, and the rate at which these substrates were used. Soil enzyme assays revealed greater laccase, peroxidase, and cellulase activities in soils associated with infected trees. Thus, event cascades associated with severe dwarf mistletoe infection not only significantly affected soil fungal species composition and increased species diversity, but also impacted on carbon-related function and functional diversity. Given the geographic range of this pathogen, and forecasts that epidemics of this disease will increase in range in severity with global climate change, these effects have the potential to significantly impact local and global carbon budgets.

Diversity of soil basidiomycete communities associated with Quercus suber L. in Portuguese montados

The montado is an agro-silvo pastoral system characterized by an open oak formation combined with shrubs or a rotation of cultures/pastures/fallow. A key, but frequently overlooked, component of these systems is the fungal community associated with the dominant oaks. We present a study of the basidiomycete community in montado areas of Quercus suber L. from Central-Western Portugal, based on fruiting body assessment and PCR-DGGE of soil mycelium. Soil available phosphorus and organic matter content were also determined. The most frequent fruiting body found in the cork oak montados belonged to Lactarius, Clitocybe, Russula and Cortinarius species. Lactarius chrysorrheus was the most widely distributed species. Soil management practices cause a temporal increase in soil available phosphorus, and harrowing and fertilizer application also result in an increase in soil organic matter content. Mechanical clearing with recent soil disturbance had a negative effect on the richness and diversity of the basidiomycete community assessed using fruiting body. The molecular analysis of the basidiomycete community separated the sites with recent shrub-removal from the others sites. Our results show that soil management techniques that avoid disturbance of the top-soil layers are the best way to preserve the structure and diversity of the soil fungal community in the montado.

Effect of continuous olive mill wastewater applications, in the presence and absence of nitrogen fertilization, on the structure of rhizosphere-soil fungal communities

Olive mill wastewater (OMW) is rich in potentially toxic organics precluding its disposal into water receptors. However, land application of diluted OMW may result in safe disposal and fertilization. In order to investigate the effects of OMW on the structure of soil fungal groups, OMW was applied daily to pepper plants growing in a loamy sand and a sandy loam at two doses for a period of 3 months (total OMW equivalents 900 and 1800 m(3) ha(-1)). Nitrogen (N) fertilization alleviated N scarcity and considerably enhanced plant biomass production; however, when applied in combination with the high OMW dose, it induced plant stress. OMW applications resulted in marked changes in the denaturing gradient gel electrophoresis patterns of soil basidiomycete communities, while concurrent N fertilization reduced these effects. In contrast, the ascomycete communities required N fertilization to respond to OMW addition. Cloning libraries for the basidiomycete communities showed that Cryptococcus yeasts and Ceratobasidium spp. dominated in the samples treated with OMW. In contrast, certain plant pathogenic basidiomycetes such as Thanatephorus cucumeris and Athelia rolfsii were suppressed. The observed changes may be reasonably explained by the capacity of OMW to enrich soils in organic substrates, to induce N immobilization and to directly introduce OMW-derived basidiomycetous yeasts.

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

Abstract 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.

Basidiomycete fungal communities in Australian sclerophyll forest soil are altered by repeated prescribed burning

Soil basidiomycetes play key roles in forest nutrient and carbon cycling processes, yet the diversity and structure of below ground basidiomycete communities remain poorly understood. Prescribed burning is a commonly used forest management practice and there is evidence that single fire events can have an impact on soil fungal communities but little is known about the effects of repeated prescribed burning. We have used internal transcribed spacer (ITS) terminal restriction fragment length polymorphism (T-RFLP) analysis to investigate the impacts of repeated prescribed burning every two or four years over a period of 30 years on soil basidiomycete communities in an Australian wet sclerophyll forest. Detrended correspondence analysis of ITS T-RFLP profiles separated basidiomycete communities in unburned control plots from those in burned plots, with those burned every two years being the most different from controls. Burning had no effect on basidiomycete species richness, thus these differences appear to be due to changes in community structure. Basidiomycete communities in the unburned control plots were vertically stratified in the upper 20 cm of soil, but no evidence was found for stratification in the burned plots, suggesting that repeated prescribed burning results in more uniform basidiomycete communities. Overall, the results demonstrate that repeated prescribed burning alters soil basidiomycete communities, with the effect being greater with more frequent burning.

Molecular diversity of soil basidiomycete communities in northern-central New South Wales, Australia

Basidiomycete communities were profiled using terminal RFLP (TRFLP) and amplified ribosomal DNA restriction analysis (ARDRA) approaches at seven field sites under differing land use in northern-central New South Wales (NSW), Australia. TRFLP data indicated greater basidiomycete species richness at sites with natural vegetation. Sixty-seven basidiomycete ARDRA-types were detected. Various putatively ectomycorrhizal fungi were detected at all sites with native vegetation. Most ectomycorrhizal taxa had affinities to the genus Tomentella, while two Pisolithus taxa and putatively ectomycorrhizal Cantharellales taxa were also detected. Although soils under woodland or grassland communities supported a range of putatively saprotrophic taxa, only members of the Ceratobasidiales were detected in soils under agricultural land use. This study is the first investigation of fungal communities in soils of northern-central NSW, Australia.

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.

Biodegradation of hexachlorobenzene by basidiomycetes in soil contaminated with industrial residues

Hexachlorobenzene (HCB), one of twelve compounds classified as ‘persistent organic pollutants’ (POP), is a byproduct of the manufacture of organochlorine compounds, and is a cause of environmental contamination in several parts of the world. Its degradation by Brazilian basidiomycetes was studied through chromatographic analyses and monitoring of the production of 14CO2 from [14C]HCB in the soil. Nineteen strains of basidiomycetes were found to be capable of tolerating concentrations of 5000 to 50,000 mg of HCB kg−1 of soil. In spite of the low rates of production of 14CO2, Psilocybe cf. castanella CCB444 and Lentinus cf. zeyheri CCB274 were capable of removing nearly 3150 and 1400 mg of HCB kg−1 from respective soil samples, during a 65-day study period.

Isolation of saprophytic basidiomycetes from soil

A method with the combined advantages of soil particle washing, selective inhibitors, and an indicator substrate was developed to isolate saprophytic basidiomycetes from soil. Organic particles were washed from soil and plated on a medium containing lignin, guaiacol, and benomyl, which reduced mold growth and allowed detection of basidiomycetes producing laccase or peroxidase. The 64 soil samples yielded 67 basidiomycete isolates, representing 51 groups on the basis of morphology and physiology. This method should facilitate investigations into the biodiversity of soil basidiomycetes and yield organisms that are useful in bioremediation of soils contaminated with pesticides or other recalcitrant aromatic compounds.

Role of soil and residue microorganisms in determining the extent of residue decomposition in soil

The effects of residue (wheat straw or sewage-sludge compost) incorporation in soil and the relative contribution of microorganisms in the residues, or in the soil to decomposition of the added residue, (CO 2 production) was evaluated in an incubation experiment. All residues and soils were adjusted to 33 kPa moisture tension and maintained at 25°C under a constant flow of CO 2-free air for 72 days. Residue decomposition was determined by monitoring CO 2 evolution from the treatments. Mixing an aged sewage-sludge compost (10%, 224 Mg ha −1) with soil stimulated decomposition of the compost 1.64-fold when compared with any of the localized placements, and indicated that the indigenous soil microorganisms were the major contributors to the transformations of this mature compost. Wheat straw was populated with organisms capable of decomposing readily-available substrates in the straw during the first stage of the decomposition, whereas it appeared that soil organisms contributed to an acceleration of straw decomposition during the final stages. After 65 days approx. 30% of the added wheat straw C had been evolved as CO 2. Soil basidiomycetes doubled the extent of decomposition when the indigenous decomposers in wheat were inactivated by γ-irradiation. Model equations are presented for residue decomposition relative to time.

The effect of water potential on the growth of some soil basidiomycetes

The effect of water potential on the growth of some soil basidiomycetes

Studies on Basidiomycetes in soil

The isolation of Basidiomycetes from soil has been studied. While extensive series of dilution and soil plates from wheat-field and pasture soil failed to record these fungi, they were isolated from roots, and from hyphae, rhizomorphs, and sclerotia picked out from soil. Over a 3-year period, no basidiomycete fructifications were found in the wheat-field although isolations from soil and roots showed that the field had an abundant and varied population of Basidiomycetes. While fructifications were obtained from the pasture, the species thus seen fruiting were different from those isolated from soil and roots, showing that the population was more varied than a study of fructifications alone would suggest. Some Basidiomycetes have been induced to fruit in culture.