Scanning electron microscopic study of vegetative mycelium of higherBasidiomycetes

The morphological character of vegetative mycelium was studied in eight species of higherBasidiomycetes by scanning electron microscopy. Morphology and variability of anastomoses, hyphal outgrowths, ornamentation and exudates, together with other irregularities in the form of hyphae are described and discussed from the point of view of their possible significance in the culture identification and taxonomic exploitation at the species level. Some of the morphological structures as observed in the scanning electron microscope are described here for the first time.     

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.     

Wirtelschnallen bei homo- und heterothallischenCoprinus-Arten (Agaricales)

Within agaric fungi whorled (multiple) clamps have been described so far only for the homothallicCoprinus myceliocephalus Lange. The new speciesCoprinus verticillatus Schulz-Weddigen (ined.) develops fertile mycelia which form septae without or with 1–5 clamps. Stronger hyphae tend to branch dichotomously. Most cells of the vegetative system contain many nuclei. Though single spore cultures remain sterile, they can develop multiple clamps, too. Moreover, very young mycelia produce a special type of oidiophores. According to its mating interactions a bipolar pattern characterizes the heterothallicC. verticillatus.

     

Arbuscular mycorrhizal structure and fungi associated with mosses

We investigated the colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with 24 moss species belonging to 16 families in China. AM fungal structures, i.e. spores, vesicles, hyphal coils (including intracellular hyphae), or intercellular nonseptate hyphae, were found in 21 moss species. AM fungal structures (vesicles, hyphal coils, and intercellular nonseptate hyphae) were present in tissues of 14 moss species, and spores and nonseptate hyphae on the surface of gametophytes occurred in 15 species. AM fungal structures were present in 11 of the 12 saxicolous moss species and in six of the ten terricolous moss species, but absent in two epixylous moss species. AM fungal structures were only observed in moss stem and leaf tissues, but not in rhizoids. A total of 15 AM fungal taxa were isolated based on trap culture with clover, using 13 moss species as inocula. Of these AM fungi, 11 belonged to Glomus, two to Acaulospora, one to Gigaspora, and one to Paraglomus. Our results suggest that AM fungal structures commonly occur in most mosses and that diverse AM fungi, particularly Glomus species, are associated with mosses.     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

Fruit-body production and mycelial growth ofTephrocybe tesquorum in urea-treated forest soil

Tephrocybe tesquorum is an ammonia fungus that forms reproductive structures successively on the forest floor after treatment of the soil with nitrogenous materials such as aqua ammonia and urea. Forest soil was treated with urea at the rates of 0, 5, 10 and 20 mg/g fresh soil for 5 d in the laboratory, then sterilized by gamma-irradiation. Vegetative hyphae ofT. tesquorum were inoculated into the sterilized soil, and the number and weight of fruit-bodies formed and the length of vegetative hyphae, were measured for 20 d after the inoculation. Only in the urea-treated soil did this fungus produce vegetative hyphae and fruit-bodies. Fruiting started 4 to 6 d after inoculation. The weight of fruit-bodies and the length of vegetative hyphae increased with the increase in the amount of urea added. These results indicate thatT. tesquorum develops vegetative hyphae and fruit-bodies when ammonium concentration, is high in soil.     

STRUCTURE AND DEVELOPMENT OF CLEISTOIODOPHANUS CONGLUTINATUS GEN. & SP. N. (ASCOBOLACEAE)

Cleistoiodophanus represents a new coprophilous genus of the tribe Iodophaneae in the Ascobolaceae (Pezizales). The only species thus far discovered, C. conglutinatus, is described and illustrated. Aspects of its cytological development are described from cultures obtained from apothecia found on sheep dung near Gainesville, Florida. Plasmogamy occurs in acogonial coils, two or three cells of which give rise to ascogenous hyphae. Ascogonia are quickly enclosed by vegetative hyphae and the ascocarp continues in a cleistohymenial development. Unlike Iodophanus and related genera, the excipulum remains intact even after spore maturation and the asci push through the epihymenial regions to release spores. The asci are characteristically thickened at their apices, diffusely amyloid, and somewhat saccate. The asci are predominantly 8-spored, but have been found with four or 16 spores per ascus. A previously undescribed Oedocephalum imperfect stage was induced in culture.     

Anatomy and ultrastructure of mycorrhizal associations of neotropical Ericaceae

Ericaceae are obligatory associated with symbiotic fungi forming several, distinctive categories of mycorrhizas. While ericoid, arbutoid, and monotropoid mycorrhizas are known since many years from ericads of the northern hemisphere and the ericoid mycorrhiza also from Australia, a further mycorrhizal category with hyphal sheath, Hartig net, and intracellular colonization was described by us recently and termed cavendishioid mycorrhiza because it was found on Cavendishia nobilis, a species belonging to the Andean clade (Vaccinioideae) of Ericaceae. As the previous findings indicated a correlation between the mycorrhizal category and the systematic position of Ericaceae, we tested the hypothesis that other ericads of the Andean clade might also form cavendishioid mycorrhizas, while ericads occurring in the same area but not belonging to the Andean clade might not. Mycorrhizas of 20 different ericaceous species, 15 belonging to the Andean clade and 5 to other Vaccinioideae or Ericoideae, were sampled in the tropical mountain rain forest area of South Ecuador and investigated by light and electron microscopy. All the 15 members of the Andean clade ericads displayed a hyphal sheath, as well as inter- and intracellular colonization by hyphae as was found on Cavendishia previously. The five species not belonging to the Andean clade ericads displayed only intracellular colonization by hyphae and hence were typical ericoid mycorrhizal. Ultrastructural studies revealed Sebacinales and ascomycetes as mycorrhiza formers in both associations even within one single cell. The results thus support the hypothesis that the Andean clade of Ericaceae forms mycorrhizas distinct from the arbutoid category and most likely presents an independent evolutionary line in the Ericaceae derived from the ericoid mycorrhizas, justifying the new term “cavendishioid mycorrhiza”.     

Fungal biodiversity in aquatic habitats

Fungal biodiversity in freshwater, brackish and marine habitats was estimated based on reports in the literature. The taxonomic groups treated were those with species commonly found on submerged substrates in aquatic habitats: Ascomycetes (exclusive of yeasts), Basidiomycetes, Chytridiomycetes, and the non-fungal Saprolegniales in the Class Oomycetes. Based on presence/absence data for a large number and variety of aquatic habitats, about 3,000 fungal species and 138 saprolegnialean species have been reported from aquatic habitats. The greatest number of taxa comprise the Ascomycetes, including mitosporic taxa, and Chytridiomycetes. Taxa of Basidiomycetes are, for the most part, excluded from aquatic habitats. The greatest biodiversity for all groups occurs in temperate areas, followed by Asian tropical areas. This pattern may be an artifact of the location of most of the sampling effort. The least sampled geographic areas include Africa, Australia, China, South America and boreal and tropical regions worldwide. Some species overlap occurs among terrestrial and freshwater taxa but little species overlap occurs among freshwater and marine taxa. We predict that many species remain to be discovered in aquatic habitats given the few taxonomic specialists studying these fungi, the few substrate types studied intensively, and the vast geographical area not yet sampled.     

Paradiplozoon homoion Bychowsky & Nagibina, 1959 versus P. gracile Reichenbach-Klinke, 1961 (Monogenea): two species or phenotypic plasticity?

Specimens of the Paradiplozoon homoion-complex were collected from ten species of cyprinid fish in the Czech Republic. A combined molecular and morphometric approach was performed to distinguish Paradiplozoon homoion and P. gracile. The second internal transcribed spacer (ITS2) of the ribosomal RNA genes was amplified and sequences were analysed. No variability in the analysed sequences was detected. Measurements of clamps and the central hooks obtained from specimens from different host species were compared. Great variability was found in the length and width of the third pair of clamps. No significant differences were detected in the measurements of the central hook sickle. A positive relationship was found between host size and each of the following measurements of the third pair clamps: length and width of the whole clamp; and length of the median plate of the third pair of clamps. The length of the median plate of the attachment clamps may be a useful character for species identification of diplozoids. Further molecular and morphometric studies are required to resolve this taxonomic problem and, henceforth, we suggest considering P. gracile as a species inquirenda.     

Genetic Diversity of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

Free-Radical Scavenging Activity of Submerged Mycelium Extracts from Higher Basidiomycetes Mushrooms

Twenty-four Basidiomycetes strains were evaluated to determine their free-radical scavenging capacity in submerged cultivation. The scavenging capacity of the extracts varied from 1 to 85% depending on the mushroom species, solvent used, and concentration. A calculation of EC₅₀ of extracts from several wood-rotting Basidiomycetes showed high scavenging abilities at low effective concentration.     

A predatory fungus (Hyphomycetes: <Emphasis Type="Italic">Lecophagus</Emphasis>) attacking Rotifera and Tardigrada in maritime Antarctic lakes

Lecophagus antarcticus, described as a new species of predaceous Hyphomycete of rotifers and tardigrades, was collected from cyanobacterial mats and sediments at shallow lake margins on Signy Island, South Orkney Islands, Antarctica. The new species differs from similar Lecophagus species by larger, more robust vegetative hyphae and by producing septate conidiophore branches or conidiophores as terminals of lateral vegetative hyphae. This is the first report of this genus from either freshwater or polar regions.     

Sistotrema is a genus with ectomycorrhizal species − confirmation of what sequence studies already suggested

The ectomycorrhizal status of Sistotrema sp. is shown by morphological–anatomical and molecular identification, confirming earlier reports about Sistotrema DNA in ectomycorrhizae (ECM). For molecular identification of the ECM nuclear rDNA ITS sequences obtained from mycorrhizal root tips and fruitbodies of Sistotrema sp. were compared. Blast searches using the Sistotrema sp. sequences as query were performed in GenBank and UNITE for comparison with previously published Sistotrema sequences. The morphological–anatomical characterization of the ECM used well-established protocols for the examination of all mantle parts and rhizomorphs in different sections and views including detailed illustrations. The ECM are irregularly monopodial–pyramidal, whitish ochre to yellow ochre, and woolly. Older ones become more greyish and silvery at some patches. Diagnostic anatomical characteristics are irregularly inflated emanating and rhizomorph hyphae, ampullately inflated clamps, and the occurrence of yellow drops within the hyphae. The plectenchymatous mantle shows ring-like arranged hyphae, and a slightly gelatinous matrix. The ECM of Sistotrema sp. are compared to those of other species that form distinctly ampullate hyphae in rhizomorphs, too. The anatomically most similar ECM to those of this Sistotrema specimen are those of Hydnum repandum.     

Morphology, anatomy, and molecular studies of the ectomycorrhiza formed axenically by the fungus Sistotrema sp. (Basidiomycota)

Several species of the corticioid (resupinate) genus Sistotrema of the cantharelloid clade (Basidiomycota) were recently found to be ectomycorrhizal. This changed the traditional assertion that all Sistotrema species are strictly wood rotting and suggests that the genus may be polyphyletic. In the present investigation, a still unknown root tip-associated fungal specimen (EW63) was isolated and found to be associated with an above-ground fruiting body. Sequencing of the ITS and the nucLSU DNA regions and phylogenetic analyses verified that the root-associated fungus and the fruiting body represented the same species, which was found to belong to the genus Sistotrema. To prove the ectomycorrhizal status of this strain, axenic Pinus sylvestris resyntheses in flask cultures were conducted. Growth parameters of the seedlings were determined and the morphology and anatomy of the synthesized mycorrhizas were described. Length and dry mass of the Pinus shoot as well as those of the total root tips were found to be enhanced as a result of the mycorrhizal association. Mycorrhizal frequency was high (51.5%) in these cultures. Mycorrhizal root tips were cottony light ochre with a thin plectenchymatic hyphal mantle. The clamps of the fruiting body hyphae as well as the mycorrhiza were ampullately inflated. This is the first report proving in axenic culture that a fungus belonging to the genus Sistotrema forms true ectomycorrhiza.     

The ectomycorrhiza <Emphasis Type="Italic">Piceirhiza internicrassihyphis</Emphasis>: A weak competitor of <Emphasis Type="Italic">Cortinarius obtusus</Emphasis>?

The ectomycorrhiza (ECM) Piceirhiza internicrassihyphis on Picea abies is described in detail. It is yellowish brown to brown with a mantle of a transitional type between plectenchymatous and pseudoparenchymatous. The inner mantle layers show some thick-walled hyphae, often with a few of them growing in parallel. These thick-walled hyphae contain particles which turn brownish in Melzer’s reagent, and their septa and walls are partially amyloid. Rhizomorphs occur infrequently and are undifferentiated, and are composed of rather loosely woven hyphae of uniform diameter. This combination of characteristics has not yet been identified in any other ECM. The lack of cystidia and the presence of amyloidy suggest that this ECM is formed by a species of the family Thelephoraceae. Piceirhiza internicrassihyphis can be found associated with ECM of Cortinarius obtusus, and the hyphae of the latter species often grow on the surface of P. internicrassihyphis and can even cover the whole tip. Although there is a close association and possibly a penetration between these two fungi, it can be excluded that the thick-walled hyphae of the inner mantle layers originate from C. obtusus. Some emanating hyphae of C. obtusus ECM seem to interact with those of P. internicrassihyphis, as indicated by short, hyphal outgrowths which were found attached to the foreign hyphae.     

Ganoderma carocalcareus sp. nov., with crumbly-friable context parasite to saprobe on Anthocleista nobilis and its phylogenetic relationship in G. resinaceum group

A new species Ganoderma carocalcareus (Basidiomycota, Ganodermataceae) was collected on living trunk and dead stumps of Anthocleista nobilis (Gentianaceae) in waterlogged swamps in the Mbalmayo Forest Reserve, Cameroon, and identified on the basis of morphology and phylogenetic analyses inferred from mitochondrial small subunit (mtSSU) and internal transcribed spacer (ITS1-5.8S-ITS2) rDNA sequences. Distinct phenotypic characteristics of the new species are dimorphism of basidiomata and variability in context structure and texture over developmental stages. The young basidiomata is ungulate to punk-shaped with context composed of vegetative hyphae attended by scattered, orbicular, smooth, thick-walled chlamydospores, and the mature basidiomata is cushion- to bracket-like with context entirely consisting of chlamydospores masses. This ontogeny intimates the origin of chlamydospores, for which the biogenesis correlates the vanishing of vegetative hyphae throughout the basidiomata maturation. Morphological comparison included Tomophagus colossus (=G. colossus), G. subamboinense and G. weberianum, the known Ganodermataceae species producing chlamydospores and or gasterospores in basidiomata tissues, and G. resinaceum, the closest species with regard to morphology. It followed that G. carocalcareus could not be assigned to these or any other known Ganoderma species. Analyses of mtSSU and ITS rDNA sequence data resolved G. carocalcareus in the G. resinaceum group as a distinct species, but being a close relative of both G. subamboinense and G. weberianum.     

Aphanomyces frigidophilus sp. nov. from eggs of Japanese char,Salvelinus leucomaenis

Aphanomyces frigidophilus sp. nov. was obtained from eggs of Japanese char,Salvelinus leucomaenis, from Tochigi Prefectural Fisheries Experimental Station, Utsunomiya, Japan. Vegetative hyphae were delicate, slightly wavy, moderately branched. Zoosporangia were isodiametric with the vegetative hyphae. Oogonia were abundant, originating on short stalks from lateral sides of hyphae. Oogonia were spherical, subspherical or pyriform, with a single subcentric oospore inside. Outer surfaces of oogonia were roughened with short papillate, crenulate or irregular ornaments. Antheridia and oospore germination were not observed. Zoospore germination and vegetative growth were found from pH 5.0 to 11.0. Zoospore production was highest at 10°C, whereas rapid growth occurred at 20–25°C. Vegetative growth of the fungus declined from the maximal level at 25°C to less than half maximal at 30°C and completely disappeared at 35°C.