A PCR/RFLP technique to characterize fungal species in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> Hill ex. Maiden ectomycorrhizas

With the increasing awareness of the significance of mycorrhizas, research is focusing on studies to elucidate the contribution of the symbiosis to ecosystem dynamics. In this sense, molecular biology has acquired great significance. PCR/RFLP techniques were adapted to characterize ectomycorrhizal fungi associated with Eucalyptus grandis.The ITS region of the fungal rDNA from pure cultures and from of mycorrhizas synthesized in vitro was amplified. Primers NSA3/NLC2 were used followed by a nested reaction with primers ITS1F/NLB3. Amplicons were then digested with the enzymes MboI, HinfI and TaqI. Amplification resulted in a 1,000-bp fragment for basidiomycetes and a 1,500 bp fragment for Cenococcum geophillum (an ascomycete). There was no amplification of the plant DNA. The enzymes MboI and HinfI were more effective than TaqI, resulting in patterns of two to five fragments allowing the identification of the isolates both in culture and in mycorrhizas. HinfI allowed greater differentiation among the isolates and a higher number of polymorphisms. Restriction with TaqI resulted in too many fragments. Amplification efficiency for the fungal DNA was 64% in culture and 87% in mycorrhizas. The modified methodology represents a valuable tool to complement traditional approaches in ecosystem studies.     

秦岭辛家山林区锐齿栎外生菌根真菌多样性

以秦岭辛家山林区锐齿栎为研究对象,通过野外调查结合形态学和分子生物学方法,观察和鉴定与其共生的外生菌根真菌。结果确定了3种子囊菌和48种担子菌,分属于10科14属,其中毛革菌属Tomentella是优势类群,丝伞盖属1 Inocybe 1、Russula persicina、毛革菌属1 Tomentella 1、毛革菌属2 Tomentella 2、块菌属Tuber、绒盖牛肝菌属Xerocomus是常见种,其余都为稀有种。两样地锐齿栎外生菌根真菌除丰富度指数外,Simpson指数、Shannon指数以及Pielou均匀度指数差异不显著;Jaccard相似性指数和Sorenson相似性指数分别为0.1154和0.2069。     

Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient

Ericaceous dwarf shrubs including Calluna vulgaris and Vaccinium spp. occur both in open heathland communities and in forest ecosystems as understory vegetation. Ericaceous shrubs were once thought to form ericoid mycorrhizal associations with a relatively narrow range of ascomycetous fungi closely related to, and including, Rhizoscyphus ericae. However, perceptions have recently changed since the realization that a broader range of ascomycete fungi, and in some cases basidiomycete fungi, can also form associations with the roots of ericaceous plants. We used a combination of molecular approaches, including denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, cloning and sequencing, to investigate the diversity of fungi associated with C. vulgaris roots collected across a heathland/native Scots pine forest vegetation gradient. We also determined differences in fungal community composition between roots of co-occurring C. vulgaris and Vaccinium myrtillus in the forest understory. Collectively, the data show that a large diversity of potentially ericoid mycorrhizal fungal taxa associate with roots of C. vulgaris and V. myrtillus, and that ascomycetes were about 2.5 times more frequent than basidiomycetes. The assemblages of fungi associated with C. vulgaris and V. myrtillus were different. In addition, the community of fungi associated with C. vulgaris hair roots was different for samples collected from the forest, open heathland and a transition zone between the two. This separation was partly, but not entirely, due to the occurrence of typical ectomycorrhizal basidiomycetes associated with the hair roots of C. vulgaris in the forest understory. These data demonstrate that forest understory ericaceous shrubs associate with a diverse range of ascomycete and basidiomycete taxa, including typical ectomycorrhizal basidiomycetes.     

中国菌根食用菌名录

菌根食用菌是一个未被合理利用的非木质林产品类群.通过文献和数据调研,本名录共收录532个中国菌根食用菌分类单元,它们隶属于28科62属,包括子囊菌门的39个分类单元以及担子菌门的493个分类单元.其中红菇科和牛肝菌科种类最丰富,红菇属、乳菇属、枝瑚菌属、蜡伞属、乳牛肝菌属、口蘑属、块菌属、牛肝菌属、鹅膏菌属和丝膜菌属是...     

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.     

ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.     

Multiple gene genealogies and species recognition in the ectomycorrhizal fungus Paxillus involutus

Paxillus involutus (basidiomycetes, Boletales) is a common ectomycorrhizal fungus in the Northern Hemisphere. The fungus displays significant variation in phenotypic characters related to morphology, physiology, and ecology. Previous studies have shown that P. involutus contains several intersterility groups and morphological species. In this study, we have used concordance of multiple gene genealogies to identify genetically isolated species of P. involutus. Fragments from five protein coding genes in 50 isolates of P. involutus collected from different hosts and environments in Europe and one location in Canada were analysed using phylogenetic methods. Concordance of the five gene genealogies showed that P. involutus comprises at least four distinct phylogenetic lineages: phylogenetic species I (with nine isolates), II (33 isolates), III (three isolates), and IV (five isolates). The branches separating the four species were long and well supported compared with the species internodes. A low level of shared polymorphisms was observed among the four lineages indicating a long time since the genetic isolation began. Three of the phylospecies corresponded to earlier identified morphological species: I to P. obscurosporus, II to P. involutus s. str., and III to P. validus. The phylogenetic species had an overlapping geographical distribution. Species I and II differed partly in habitat and host preferences.     

Fungal associates of <Emphasis Type="Italic">Pyrola rotundifolia</Emphasis>, a mixotrophic Ericaceae,from two Estonian boreal forests

Pyrola rotundifolia (Ericaceae, Pyroleae tribe) is an understorey subshrub that was recently demonstrated to receive considerable amount of carbon from its fungal mycorrhizal associates. So far, little is known of the identity of these fungi and the mycorrhizal anatomy in the Pyroleae. Using 140 mycorrhizal root fragments collected from two Estonian boreal forests already studied in the context of mixotrophic Ericaceae in sequence analysis of the ribosomal DNA internal transcribed spacer region, we recovered 71 sequences that corresponded to 45 putative species in 19 fungal genera. The identified fungi were mainly ectomycorrhizal basidiomycetes, including Tomentella, Cortinarius, Russula, Hebeloma, as well as some ectomycorrhizal and/or endophytic ascomycetes. The P. rotundifolia fungal communities of the two forests did not differ significantly in terms of species richness, diversity and nutritional mode. The relatively high diversity retrieved suggests that P. rotundifolia does not have a strict preference for any fungal taxa. Anatomical analyses showed typical arbutoid mycorrhizae, with variable mantle structures, uniseriate Hartig nets and intracellular hyphal coils in the large epidermal cells. Whenever compared, fungal ultrastructure was congruent with the molecular identification. Similarly to other mixotrophic and autotrophic pyroloids in the same forests, P. rotundifolia shares its mycorrhizal fungal associates with surrounding trees that are likely a carbon source for pyroloids.     

Survey of ectomycorrhizal,litter-degrading,and wood-degrading <Emphasis Type="Italic">Basidiomycetes</Emphasis> for dye decolorization and ligninolytic enzyme activity

Basidiomycetes are essential in forest ecology, being deeply involved in wood and litter decomposition, humification, and mineralization of soil organic matter. The fungal oxidoreductases involved in these processes are today the focus of much attention with a view to their applications. The ecological role and potential biotechnological applications of 300 isolates of Basidiomycetes were assessed, taking into account the degradation of model dyes in different culture conditions and the production of oxidoreductase enzymes. The tested isolates belong to different ecophysiological groups (wood-degrading, litter-degrading, ectomycorrhizal, and coprophilous fungi) and represent a broad systematic and functional biodiversity among Basidiomycetes occurring in deciduous and evergreen forests of northwest Italy (Piedmont Region). The high number of species tested and the use of different culture conditions allowed the investigation of the degradation activity of several novel species, neglected to date. Oxidative enzyme activities varied widely among all ecophysiological groups and laccases were the most commonly detected enzymes. A large number of isolates (86%), belonging to all ecophysiological groups, were found to be active against at least one model dye; the wood-degrading fungi represented the most efficient group. Noteworthily, also some isolates of litter-degrading and ectomycorrhizal fungi achieved good decolorization yield. The 25 best isolates were then tested against nine industrial dyes commonly employed in textile industries. Three isolates of Bjerkandera adusta efficiently decolorized the dyes on all media and can be considered important candidates for application in textile wastewater treatment.     

Effects of isolates of ectomycorrhizal fungi and endophytic Mycelium radicis atrovirens that were dominant in soil from disturbed sites on growth of Betula platyphylla var. japonica seedlings

Effects of ectomycorrhizal fungi and endophytic Mycelium radicis atrovirens Melin (MRA) on growth of Betula platyphylla var. japonica seedlings were investigated under aseptic culture conditions. Three isolates of ectomycorrhizal fungi and two isolates of MRA were used. One MRA isolate was Phialocephala fortinii. Previous field work revealed that these isolates were dominant on the roots of B. platyphylla var. japonica seedlings grown in a mineral subsoil that had been exposed by the removal of surface soil. After a 100-day incubation, the growth of the seedlings was significantly enhanced by the colonization of these ectomycorrhizal fungal isolates as compared with uninoculated seedlings. In contrast, the growth of seedlings was retarded by the colonization of the MRA isolates. The growth of seedlings that were co-inoculated with ectomycorrhizal fungi and MRA was similar to that of uninoculated seedlings in most cases. These results suggest that ectomycorrhizal fungi have a beneficial effect on the growth of B. platyphylla var. japonica seedlings and that they suppress the deleterious effect of MRA. Thus, these ectomycorrhizal fungi probably have an important role in establishing B. platyphylla var. japonica seedlings during the initial stage of re-vegetation following site disturbance by the removal of surface soil.     

Identification of ectomycorrhizae formed betweenTricholoma matsutake andPinus densiflora by polymerase chain reaction (PCR) targeting retroelement coding regions

We previously reported that conservation and diversification of repetitive sequences carrying motifs of retroposons have occurred inTricholoma matsutake and related ectomycorrhizal basidiomycetes through their evolution. Here we report that the polymerase chain reaction using primers designed to amplify retroelement coding regions specified ectomycorrhizae formed betweenT. matsutake andPinus densiflora.     

Screening of basidiomycetes for antimicrobial activities

As a part of a screening programme developed to evaluate the antimicrobial activity of basidiomycetes, 317 isolates representing 204 species collected in Spain were screened against a range of human clinical pathogens and laboratory controls. Extracts from 45% of the isolates, representing 109 species, showed antimicrobial activity. Antibacterial activity was more pronounced than antifungal activity. The proportion of extracts from basidiomycetes showing antimicrobial activity was similar to or above that obtained for representative orders of Ascomycetes, such as Pezizales and Xylariales, but lower than that produced by members of the orders Diaporthales, Eurotiales, Hypocreales, Leotiales and Sordariales. Suprageneric taxa (orders and families) did not show pronounced differences in their antimicrobial activities though such differences were observed at the genus level, suggesting that the ability to produce these bioactive compounds is not homogenously distributed amongst the basidiomycetes. Isolates from some species showed large differences in their ability to produce metabolites with antimicrobial activity, possibly reflecting genetic differences at the infraspecific level.     

Roles of diverse fungi in larch needle-litter decomposition

Functional biodiversity of fungi in larch (Larix leptolepis) forests needle-litter decomposition was examined by a pure-culture test. Weight loss of larch-needle litter, utilization pattern of lignocellulose and chemical composition of remaining litter were investigated and compared for 31 isolates in 27 species of basidiomycetes and ascomycetes. Weight loss (% original weight) of litter ranged from -2.0% to 14.2%. Mean weight loss of litter caused by the basidiomycetes was not significantly different from that caused by the ascomycetes. Basidiomycetes caused loss of lignin and carbohydrates in variable proportions, while ascomycetes exclusively attacked carbohydrates without delignification. The content of lignin and nitrogen in remaining litter was not significantly correlated when both basidiomycetes and ascomycetes were included. However, the correlation coefficient was significant when the relationship was examined separately for basidiomycetes, indicating that the degree of selective delignification determined the final nitrogen content in litter. Possible effects of fungal colonization on needle-litter decomposition in larch forests are discussed.     

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.     

Degradation of Soil Humic Extract by Wood- and Soil-Associated Fungi, Bacteria, and Commercial Enzymes

Abstract An alkaline humic extract (HE) of a black calcareous forest mull was exposed to 36 fungal and 9 eubacterial isolates in liquid standing culture. At 21 d in fungi, and 4 d in bacteria, the groups of wood-degrading basidiomycetes, terricolous basidiomycetes, ectomycorrhizal fungi, soil-borne microfungi, and eubacteria had reduced the absorbance (A ₃₄₀) of HE media by 57, 28, 19, 26 and 5%, respectively. Gel permeation chromatography revealed that the large humic acid molecules were more readily degraded than the smaller fulvic acid molecules and served as a sole source of carbon and energy. The more active HE degraders reduced the overall molecular weight of humic and fulvic acids by 0.25 to 0.47 kDa. They also reduced the chemical reactivity of HE to tetrazotized o-dianisidine, indicating the degradation of hydroxylated aromatic molecules (which are responsible for this reaction). Decreases in absorbance, molecular weight, and reactivity were caused by fungal manganese peroxidase, horseradish peroxidase, β-glucosidase, and abiotic oxidants such as H₂O₂ and Mn(III) acetate. It is concluded that fungi, some of which are propagated in contaminated soils to control xenobiotics, metabolize HE compounds enzymatically. They use enzymes which are also involved in the degradation of soil xenobiotics. Because of reductions in the molecular weight of HE, which is a potential carrier of heavy metal ions and xenobiotics, solubility and motility of humic substances in soil and surface waters are increased. Received: 4 March 1998; Accepted: 1 June 1998     

Detection and Identification of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplified Genes Encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

Relative importance of the endomycorrhizal and (or) ectomycorrhizal associations in Allocasuarina and Casuarina genera

This work was carried out to determine the relative importance of the endomycorrhizal and (or) ectomycorrhizal association in species of Casuarina and Allocasuarina. Under axenic conditions, Pisolithus and Scleroderma isolates formed ectomycorrhizas with a mantle and a Hartig net on Allocasuarina verticillata but failed to form a Hartig net on Casuarina glauca. In a controlled soil system, C. glauca was inoculated with the endomycorrhizal fungus Glomus intraradices Schenck & Smith, and A. verticillata was inoculated with Pisolithus albus IR100 Bougher & Smith and (or) G. intraradices. Both symbionts significantly stimulated growth in both plant species. For A. verticillata, its growth response to ectomycorrhizal inoculation was higher than to endomycorrhizal inoculation. When both symbionts were inoculated, antagonism among the fungal isolates was observed with a higher ectomycorrhizal colonization. These results showed that A. verticillata was ectomycorrhizal dependent, whereas C. glauca was endomycorrhizal dependent. From a practical point of view, this study shows the importance of selecting compatible mycorrhizal fungi for developing successful inoculation programmes. In addition, it would help to further research and determine the effect of ecto- and endo-mycorrhizal symbiosis on the formation and function of N2-fixing actinorhizal nodules.     

Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.