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.     

Die Verschiedenartigkeit von Haftorganen einiger Flechten

Summary The rhizines of the lichens, Parmelia caperata, Parmelia trichotera and Lobaria pulmonaria were studied with the Stereoscan scanning electronmicroscope and in ultrathin sections with the transmission electronmicroscope. The rhizines are composed of fungal hyphae.The fungal hyphae in the rhizines of the thallus of Parmelia caperata and in the cilia at the thallus border of Parmelia trichotera are connected by a glue-like substance. The ends of the Parmelia caperata rhizines are flattened and enlarged. With these footlike rhizines the thalli are in good connection with the substratum. The cilia at the thallus border of Parmelia trichotera have a tip by which the thallus is fixed on bark or rocks. The cell walls of the fungi hyphae in the Parmelia caperata rhizines and in the Parmelia trichotera cilia are 150–400 nm thick.The rhizines of Lobaria pulmonaria consist of fungi hyphae which are interlaced without a gluey substance. The thallus of Lobaria pulmonaria is connected to the substratum through the tips of single hyphae. The hyphae walls of Lobaria pulmonaria are 800–1800 nm thick.     

Microorganisms asssociated with Withania somnifera leaves

Microorganisms including bacteria, actinomycetes and fungi were recovered from the leaves of Withania somnifera, which were collected from two altitudinal ranges (0–300 m and 1700–2000 m) in the Asir region, Saudi Arabia. Types and numbers of microorganisms varied according to the altitude and the month of collection. The number of microorganisms was higher on old leaves than that on young ones in most cases. Low altitude exhibited more microorganisms than high altitude. The relationship between meteorological factors and type and number of the recovered microorganisms is discussed. Inoculation of detached healthy leaves of Withania by all recovered fungal species revealed only Alternaria solani as a pathogen of this plant. To confirm pathogenicity, scanning and transmission electron microscopic examination revealed the colonization of this pathogen inside the leaf tissue. Penetration of Withania leaves by the fungus occurred only through stomata, and the invading hyphae were located in the intercellular spaces of leaf tissues. Ultrastructural changes noted in infected cells included changes in chloroplasts and the invagination of the host plasma membrane.     

Diversity of arbuscular mycorrhizal fungi colonising roots of the grass species<Emphasis Type="Italic"> Agrostis capillaris</Emphasis> and<Emphasis Type="Italic"> Lolium perenne</Emphasis> in a field experiment

Analysis of arbuscular mycorrhizal (AM) fungal diversity through morphological characters of spores and intraradicular hyphae has suggested previously that preferential associations occur between plants and AM fungi. A field experiment was established to investigate whether AM fungal diversity is affected by different host plants in upland grasslands. Indigenous vegetation from plots in an unimproved pasture was replaced with monocultures of either Agrostis capillaris or Lolium perenne. Modification of the diversity of AM fungi in these plots was evaluated by analysis of partial sequences in the large subunit (LSU) ribosomal RNA (rDNA) genes. General primers for AM fungi were designed for the PCR amplification of partial sequences using DNA extracted from root tissues of A. capillaris and L. perenne. PCR products were used to construct LSU rDNA libraries. Sequencing of randomly selected clones indicated that plant roots were colonised by AM fungi belonging to the genera Glomus, Acaulospora and Scutellospora. There was a difference in the diversity of AM fungi colonising roots of A. capillaris and L. perenne that was confirmed by PCR using primers specific for each sequence group. These molecular data suggest the existence of a selection pressure of plants on AM fungal communities.     

Mites as Selective Fungal Carriers in Stored Grain Habitats

Mites are well documented as vectors of micromycetes in stored products. Since their vectoring capacity is low due to their small size, they can be serious vectors only where there is selective transfer of a high load of specific fungal species. Therefore the aim of our work was to find out whether the transfer of fungi is selective. Four kinds of stored seeds (wheat, poppy, lettuce, mustard) infested by storage mites were subjected to mycological analysis. We compared the spectrum of micromycete species isolated from different species of mites (Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae, Caloglyphus rhizoglyphoides and Cheyletus malaccensis) and various kinds of stored seeds. Fungi were separately isolated from (a) the surface of mites, (b) the mites' digestive tract (= faeces), and (c) stored seeds and were then cultivated and determined. The fungal transport via mites is selective. This conclusion is supported by (i) lower numbers of isolated fungal species from mites than from seeds; (ii) lower Shannon–Weaver diversity index in the fungal communities isolated from mites than from seeds; (iii) significant effect of mites/seeds as environmental variables on fungal presence in a redundancy analysis (RDA); (iv) differences in composition of isolated fungi between mite species shown by RDA. The results of our work support the hypothesis that mite–fungal interactions are dependent on mite species. The fungi attractive to mites seem to be dispersed more than others. The selectivity of fungal transport via mites enhances their pest importance.     

Detection of Heterobasidion annosum s. l. [(Fr.) Bref.] in Norway Spruce by Polymerase Chain Reaction

Internal transcribed spacer (ITS) sequences of the rDNA repeat unit of Heterobasidion annosum were used to design specific primers for the detection and quantification of this important forest pathogen by polymerase chain reaction (PCR). Specificity of detection was cross‐checked against a variety of other fungi (saprophytes, root pathogens, mycorrhizal fungi) which may occur in the same environment. As little as 1 pg fungal DNA (equiv. to 10–40 genomes) could be detected in 200 ng spruce root DNA (from 1 mg fresh spruce root). The Heterobasidion‐specific primers allowed simultaneous detection of Armillaria spp. in multiplex PCR. The method was successfully applied to increment cores of Norway spruce from the forest region Tharandter Wald (Saxonia, Germany), Oberbärenburg (East Ore Mountains, Saxonia) and Oberschleissheim (north of Munich, Bavaria).     

Anatomical study on the interaction between the root endophytic fungus <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis> and Chinese cabbage

Chinese cabbage roots colonized by the dematiaceous fungal taxon Heteroconium chaetospira were previously found to become highly resistant to clubroot and Verticillium yellows. The dematiaceous fungus possesses an endophytic nature, but no detailed anatomical studies on endophyte–host plant interactions have so far been provided. Light and electron microscopy revealed that hyphae of H. chaetospira were abundant on and inside the root epidermal cells by 3 weeks following inoculation. The penetration pegs easily breached into epidermal cells, and the infection hyphae penetrated into cortical cells. Some appressorium-like swollen structures formed from intracellular hyphae, but no visible degradation of the host cell walls was evident where the hyphae contacted. No visible signs of host reactions and no invagination of the host plasma membrane around the hyphae were seen in the host cells. By 8 weeks following inoculation, masses of closely packed fungal cells had been formed in some cells of the epidermis and cortical layers, but further hyphal ingress was halted, mostly in the inner cortical cell layer. Thus, root vascular cylinders remained intact.     

The occurrence of biomineralization products in four lichen species growing on sandstone in western Norway

High performance thin-layer chromatography/thin-layer chromatography, X-ray diffraction, and scanning electron microscopy analysis of thallus and lichen-rock interface samples, were undertaken to characterize biomineralization products in Fuscidea cyathoides, Ochrolechia tartarea, Ophioparma ventosa, and Pertusaria corallina, growing on sandstone in western Norway. Whewellite (monohydrate form of Ca oxalate) was found in the thallus of all species, but not in any of the weathering rinds beneath the species. A significantly higher amount of whewellite was detected in the thalli of F. cyathoides and O. ventosa than in the other two species. There were only a few differences in whewellite occurrence between the thallus edge and centre samples in the four species. HPTLC/TLC and SEM analysis indicate that lichen compounds occur within the rock beneath some of the lichen specimens. Only divaricatic acid was observed within the weathering rind beneath O. ventosa. No lichen substances were found in the weathering rind beneath F. cyathoides and P. corallina, whereas gyrophoric and lecanoric acids were found in the weathering rind beneath O. tartarea.     

Production of Polyclonal and Monoclonal Antibodies Against Hyphae from Arbuscular Mycorrhizal Fungi

Abstract

Polyclonal and monoclonal antibodies were produced against hyphae of the arbuscular mycorrhizal fungus Glomus monosporum. The polyclonal antibodies (pAbs) were raised in a rabbit by immunizing with hyphae. They were tested for their specificity by a dot-immunoblot assay (DIBA). After the third immunization, a distinct difference in the signal strength was observed between the antisera and the preimmune serum. The pAbs showed cross-reactions to a number of fungal species, both mycorrhizal and other. For the production of monoclonal antibodies (mAbs), mice were immunized intraperitoneally with hyphae. The resulting hybridoma cell culture supernatants were tested by an indirect immunolabeling procedure. For this purpose the hyphae were immobilized on silane-coated microscopic slides. The mAb 8A7 reacted with hyphae from all Glomus isolates tested so far. Cross-reactivities were not observed with hyphae from fungi of the family Acaulosporaceae, phytopathogenic fungi tested so far, or from spores from Glomus species.     

Host plant differences in arbuscular mycorrhizae: Extra radical hyphae differences between an invasive forb and a native bunchgrass

Arbuscular mycorrhizae affect grassland plant community composition and host plant nutrient uptake, and can mediate shifts in competitive outcome between plant species. Centaurea maculosa, an invasive forb from Eurasia, dominates more than 4 million hectares in the Rocky Mountain region of North America. We examined the role of AM for phosphorus (P) acquisition from a distant source for C. maculosa and Festuca idahoensis, a native bunchgrass. Plants were grown individually in pots divided by a barrier that either excluded plant roots and AM hyphae, or only plant roots. In the half of the pot without a plant, 1 of 3 P treatments was applied: no P, phosphate rock (PR) or triple superphosphate (TSP), applied at a rate of 144 mg P kg^–1 soil. After 14 weeks of growth, C. maculosa was twice as large as F. idahoensis, and neither species biomass was affected by barrier type. Phosphorus fertilizer, and especially PR, moved across the barrier to the plant side of the pot. Tissue P concentration for C. maculosa was highest with the PR treatment, and was not affected by the barrier type. In contrast, F. idahoensis tissue P concentration did not vary with barrier or P treatments. There was more AM extra radical hyphae (ERH) associated with C. maculosa than F. idahoensis, suggesting that C. maculosa provides more carbon for the AM fungi, resulting in greater ERH production, ERH soil exploration and potential for soil nutrient pool exploitation. Although not tested in this study, differences between host plants may be the result of different physiological characteristics of the host plant or differences in AM fungal species that colonize the invader, with different fungal species accessing P from different distances.     

Fungal Communities on Rock Surfaces in Demänovská Ice Cave and Demänovská Cave of Liberty (Slovakia)

This paper is the first geomycological report regarding the fungal communities on rock surfaces in the Demänovská Ice Cave and the Demänovská Cave of Liberty, Slovakia. The samples were collected in June 2014 from five locations from inside both the caves by using sterile swabs wetted with physiological saline (0.85% NaCl). The density of epilithic fungi isolated from the Demänovská Ice Cave ranged from 238.7 to 575.1 CFU (colony-forming units) per m² of the rock surface, and from the Demänovská Cave of Liberty ranged from 88.6 to 347 CFU. Seventeen different free-living culturable fungi (15 filamentous fungi, one yeast, and one yeast-like fungus) were isolated from the rock surfaces of both caves. Generally, Cladosporium cladosporioides and Aspergillus flavus were the most frequently cultured species from the Demänovská Ice Cave and the Demänovská Cave of Liberty, respectively. Free-living fungi found on the rock surfaces of both caves can lead to their slow biodegradation.     

A molecular survey of ectomycorrhizal hyphae in a California Quercus–Pinus woodland

Ectomycorrhizal (ECM) hyphal communities have not been well characterized. Furthermore, there have been few studies where the ECM hyphal community is compared to fungi detected as sporocarps or ECM-colonized root tips. We investigated fungi present as hyphae in a well-studied California Quercus–Pinus woodland. Hyphal species present were compared to those found as sporocarps and ECM root tips at the same site. Hyphae were extracted from root-restrictive nylon mesh in-growth bags buried in the soil near mature Quercus douglasii, Quercus wislizeni, and Pinus sabiniana. Taxa were identified using PCR, cloning, and DNA sequencing of internal transcribed spacer and 28s rDNA. Among the 33 species detected, rhizomorph-forming ECM fungi dominated the hyphal community, especially species of Thelephoraceae and Boletales. Most fungi in soils near Quercus spp. and P. sabiniana were ECM basidiomycetes, but we detected two ECM ascomycetes and three non-mycorrhizal fungi. Many ECM species present as hyphae were also previously detected at this site as sporocarps (18%) or on ECM root tips (58%). However, the hyphal community was mostly dominated by different taxa than either the sporocarp or ECM root communities.     

Biodegradation of ivory (natural apatite): possible involvement of fungal activity in biodeterioration of the Lewis Chessmen

Fungal biodeterioration of ivory was investigated with in vitro inoculation of samples obtained from boar and walrus tusks with the fungi Aspergillus niger and Serpula himantioides, species of known geoactive abilities. A combination of light and scanning electron microscopy together with associated analytical techniques was used to characterize fungal interactions with the ivory, including changes in ivory composition, dissolution and tunnelling, and the formation of new biominerals. The research was aimed at providing further understanding of the potential roles of fungi in the colonization and deterioration of ivory in terrestrial environments, but also contributes to our knowledge regarding the possible origins of the surface damage observed on early medieval sculptures made largely from walrus tusks, referred to as ‘the Lewis hoard of gaming pieces’, that were presumably produced for playing chess. The experiments have shown that the possibility of damage to ivory being caused by fungi is realistic. Scanning electron microscopy revealed penetration of fungal hyphae within cracks in the walrus tusk that showed also widespread tunnelling by fungal hyphae as well as ‘fungal footprints’ where the surface was etched as a consequence of mycelial colonization. Similar phenomena were observed with boar tusk ivory, while production of metabolites could lead to complete dissolution of the sample. Colonization of ivory and/or exposure to fungal activity lead to extensive secondary biomineral formation, and this was identified as calcium oxalate, mainly as the monohydrate, whewellite.     

Interactions of sterile-cultured lichen-forming ascomycetes with asbestos fibres

Sterile cultured isolates of lichen-forming ascomycetes have not yet been used to investigate mycobiont–mineral substrate interactions under controlled conditions. In this study Candelariella vitellina, Xanthoparmelia tinctina and Lecanora rupicola mycobionts were isolated and inoculated with chrysotile fibres in the laboratory, in order to verify whether physical and chemical weathering processes, which were already described in the field, may be reproduced in vitro. Tight adhesion of hyphae to chrysotile fibres was observed in all species. The adhering hyphae affected the chemical composition of asbestos fibres, with the selective depletion of magnesium being a prominent feature, as is the case in field conditions. Oxalic acid and pulvinic acid, mycobiont-derived metabolites of X. tinctina and C. vitellina, were involved in the weathering action. Time and environmental factors and the absence of biological synergisms strongly limited the chemical weathering in vitro compared with what was observed in the field. Nevertheless, the results show that in vitro incubation of sterile-cultured lichen-forming fungi with minerals is a practicable experimental system to investigate the weathering effects of different mycobionts and fungal compounds under controlled conditions.     

Extracellular protein fibrils in Chrysochromulina breviturrita (Prymnesiophyceae) and their serological relationship to fungal fimbriae

An extensive network of extracellular fibrils was revealed by negative staining in the greenish gold algal flagellate, Chrysochromulina breviturrita. These fibrils were of uniform diameter (4–5 nm), sometimes exceeding 5 m in length. In addition there were short, narrower fibrils (2–3 nm) on the surface of the flagella. Six protein bands were isolated from spent culture medium by SDS-PAGE and one of 80,000 Da was found to polymerize after dialysis into 4–5 nm fibrils identical to those found on the cell surface. Two other proteins of 58,000 Da and 65,000 Da also formed 4–5 nm fibrils but these were either rare or of a shorter length and different appearance. An antiserum directed against the surface 7 nm fibrils (fimbriae) of fungi agglutinated cells of C. breviturrita and some other Prymnesiophyceae and Chrysophyceae, but did not agglutinate cells of algal species in other groups. Immunofluorescence and protein A gold labelling confirmed that antigens related to fungal fimbriae were present on the surface of cells of C. breviturrita. Only the 80,000 and 58,000 Da proteins labelled heavily following protein A gold labelling. Some individual 4–5 nm fibrils labelled with gold were observed in the material prepared from the 80,000 Da band. These results therefore establish that C. breviturrita produces a surface network of fibrils that are serologically related to the fimbriae of fungi, and suggest a previously unrecognized relationship between members of the Prymnesiophyceae, Chrysophyceae and fungal groups.     

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 g–1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.     

Fungal specific primers for PCR‐amplification of mitochondrial LSU in lichens

Fungal specific primer sequences for the amplification of the large subunit of the mitochondrial ribosomal DNA (mtLSU) are presented in this paper. Fungal specific primers make the separation of fungal and algal cells prior to DNA‐extraction from lichens unnecessary. This is especially useful in crustose and small foliose and fruticose lichens. An example from a complex of closely related species of the crustose lichen genus Biatora shows the usefulness of mtLSU‐sequences for studies of infraspecific variability and lower level systematics of lichenized ascomycetes.     

Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR

Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga (Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex–polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores.