Tricholoma matsutake– an assessment of in situ and in vitro infection by observing cleared and stained whole roots

 Structures present within field-collected Tricholoma matsutake/Pinus densiflora ectomycorrhizas and in vitro infections of P. densiflora roots by T. matsutake were observed by clearing, bleaching and staining whole lateral roots and mycorrhizas. Field mycorrhizas were characterized by a lack of root hairs, by the presence of a sparse discontinuous mantle composed of irregularly darkly staining hyphae over the root surface, primarily behind the root cap, and by the presence of Hartig net mycelium within the root cortex. Hartig net 'palmettis' were classified into three basic structures, each with distinctive morphologies. Aerial hyphae, bearing terminal swellings, were observed emanating from the mantle. Cleared, bleached and stained in vitro-infected roots possessed multibranched hyphal structures within the host root cortex and aerial hyphae bearing terminal swellings were observed arising from the mycelium colonizing the root surface. T. matsutake on P. densiflora conforms to the accepted morphology of an ectomycorrhiza. This staining protocol is particularly suited to the study of Matsutake mycorrhizal roots and gives rapid, clear, high-contrast images using standard light microscopy while conserving spatial relationships between hyphal elements and host tissues. Accepted: 26 August 1999     

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.     

Anatomical aspects of field ectomycorrhizas on Polygonum viviparum (Polygonaceae) and Kobresia bellardii (Cyperaceae)

 Root systems of the herbaceous species Polygonum viviparum and Kobresia bellardii were excavated from an alpine site in the Rocky Mountains, Colorado, and processed for microscopic examination. Several ectomycorrhizal morphotypes were present on root systems of both species;K. bellardii often had complex clusters of mycorrhizal roots present. A mantle and Hartig net were present on all mycorrhizal root tips processed. The Hartig net was confined to the epidermis, and the parenchyma cells of this layer were radially elongated, vacuolated and contained densely staining inclusions. Intracellular hyphae and structures typical for vesicular-arbuscular mycorrhizas were never observed. Both herbaceous species, therefore, had ectomycorrhizal associations comparable to those described for woody angiosperm species. Accepted: 14 February 1998     

Competition between ectomycorrhizal fungi colonizing Pinus densiflora

 Interactive competition of Pisolithus tinctorius (Pers.) Coker et Couch with an unidentified species Tanashi 01 and Suillus luteus (L.: Fr.) S. F. Gray was investigated using a rhizobox. Pinus densiflora Sieb. et Zucc. was used as the host plant and mycelia were distinguished by hyphal color. The speed of mycelial spread differed between the fungi;P. tinctorius and Tanashi 01 grew faster than S. luteus. A P. tinctorius mycorrhizal seedling and a Tanashi 01 mycorrhizal seedling were transplanted on opposite sides of the rhizobox. The mycelia and mycorrhizae of P. tinctorius were overgrown by Tanashi 01 hyphae and development of P. tinctorius was gradually inhibited. The areas occupied by mycelia and mycorrhiza of P. tinctorius decreased by 52% and 37%, respectively, 154 days after transplantation relative to that at 91 days. In the overlap area of P. tinctorius and Tanashi 01, the latter fungus infected new root tips emerging from P. tinctorius mycorrhiza, which lacked a mantle of P. tinctorius hyphae, and formed a composite mycorrhizal structure. P. tinctorius mycorrhizae were progressively replaced by Tanashi 01 mycorrhizae. Mycelial spread of P. tinctorius and S. luteus were naturally inhibited but there was no interaction in mycorrhizal formation. Accepted: 18 June 1999     

In vitro synthesis of Pisolithus-Eucalyptus ectomycorrhizae: synchronization of lateral tip emergence and ectomycorrhizal development

 A simple and reproducible in vitro system is described for the synthesis of Pisolithus-Eucalyptus grandis ectomycorrhizae. Hyphal discs from actively growing colonies were placed in large petri dishes containing minimum nutrient agar overlaid with cellophane and allowed to grow for 7 days. Seeds were then surface sterilized and placed above the expanding fungal colonies and the plates slanted. Seedlings that germinated and grew in the presence of fungal hyphae had twice as many lateral root tips as seedlings that germinated before they were transferred onto hyphal mats. In addition, the lateral root tips of inoculated seedlings had a faster maturation rate and emerged closer to the primary root apex than non-inoculated seedlings. All lateral tips emerged in contact with fungal hyphae and the differentiation of ectomycorrhizae was followed by examining lateral tips basipetally along a single primary root. Typical ectomycorrhizae had formed on 4-day-old lateral tips, i.e. a mantle, radially elongated epidermal cells and a Hartig net commencing about 0.3 mm behind the lateral root apex. Thereafter, the mantle continued to thicken and the apical meristem diminished. The Hartig net often surrounded the apex of 11- to 12-day-old lateral root tips. This model system will facilitate detailed studies on synchronized ectomycorrhizal development and associated molecular and biochemical changes. Accepted: 12 January 1996     

Comparative studies of ectomycorrhiza formation in Alnus glutinosa and Pinus resinosa with Paxillus involutus

 Mycorrhiza ontogeny and details of Hartig net and mantle structure were compared in ectomycorrhizas synthesized in growth pouches between the broad host range fungus Paxillus involutus and the tree species European black alder (Alnus glutinosa) and red pine (Pinus resinosa). In Alnus glutinosa, a paraepidermal Hartig net was restricted to the proximal (basal) portion of first-order laterals; the hypodermal layer appeared to be a barrier to fungal penetration. Phi-thickenings were present in some cortical cells but these were not related to lack of fungal ingress into the cortex. The mantle was often present close to the root apex but in many roots it was loosely organized and patchy. In several instances, the mantle formed around the root apex was only temporary; renewed root growth occurred without the formation of a mantle. In Pinus resinosa, the Hartig net developed between cortical cell layers of monopodial and dichotomously branched first–order laterals. Fungal hyphae in the Hartig net exhibited a complex labyrinthine mode of growth. The mantle had a pseudoparenchymatous structure and covered the root, including apices of dichotomously branched roots. The Paxillus–Pinus resinosa interaction had all the characteristics of a compatible ectomycorrhizal association. The Paxillus–Alnus glutinosa interaction, however, showed only aspects of superficial ectomycorrhizas, including the presence of a minimal (sometimes absent) and mostly proximal Hartig net and variable mantle development. Sclerotia were produced in the extraradical mycelium of Paxillus involutus when associated with either Alnus glutinosa or Pinus resinosa. Accepted: 22 October 1998     

Reassociation of a modified mycorrhiza with the host plant roots (Pinus radiata) and the transfer of acetylene reduction activity

Summary Experiments were carried out to assess the potential for reassociation of modified strains of the mycorrhizal fungus Rhizopogon sp., capable of acetylene reduction activity in vitro, with the roots of its host plant (Pinus radiata). Reassociation was effected and acetylene reduction assays indicated that nitrogenase activity was present in the reassociated whole plants. Those host plants symbiotic with the modified strains had higher levels of nitrogen than those associated with the wild type fungus under nitrogen deprived conditions. Uptake of phosphate was unimpaired in the modified mycorrhiza. Electron microscopy showed that hyphae of the modified strains as well as lying in the intercellular spaces were often found within the cells of the root cortex. This was in contrast with the wild type strains where no such intracellular growth was found. One strain was sound to be pathogenic to seedlings of Pinus radiata. re]19760603     

Root Distribution and Nutrient Status of Mycorrhizal and Non-mycorrhizal Pinus sylvestris L. Seedlings Growing in a Sandy Substrate with Lignite Fragments

Reclaimed mine soils of the Lusatian mining district are characterised by small-scale heterogeneous distribution of lignite fragments of varying size embedded in a matrix of Tertiary and Quaternary sandy material. Despite amelioration with basic fly ashes, ongoing pyrite oxidation and the subsequent acidification generate a high physical and chemical heterogeneity within the substrate, which could negatively affect root proliferation. We hypothesised that this limitation for the root system may be compensated for by intensive exploration of the porous lignite fragments by roots and/or mycorrhizal hyphae to access water and nutrients stored in these fragments. To test this hypothesis, we compared growth, shoot nutrient content, and root distribution of mycorrhizal and non-mycorrhizal Pinus sylvestris L. seedlings in lignite-containing and lignite-free sandy substrate. Rhizotrons used for this experiment were filled with a sandy matrix with 6–9 evenly distributed spots of lignite fragments. Treatments included different levels of water and nutrient availability. After 8 months of growth, root tip vitality as well as growth and shoot nutrient concentration of the plants was higher for treatments with lignite spots in the sandy substrate than for sandy substrate without such amendments. Compared to the non-mycorrhizal plants, the seedlings inoculated with Paxillus involutus (Batsch) Fr. had a higher root dry mass, an increased number of root tips and a higher root length. These results confirm our hypothesis that the lignite fragments are an important nutrient and water reservoir for plants in these mine soils and they indicate that mycorrhizal colonisation may allow an intensive exploration of porous lignite fragments by mycorrhizal hyphae.     

Behaviour of the hyphae of<Emphasis Type="Italic"> Laccaria laccata</Emphasis> in the presence of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.     

Mycorrhizae in Adenostoma fasciculatum Hook. & Arn.: a combination of unusual ecto- and endo-forms

 The mycorrhizal status of Adenostoma fasciculatum, the dominant shrub in California chaparral, has been unclear. In two typical, nearly monospecificstands, A. fasciculatum was found to have arbuscules and intercellular hyphae. Antisera detected hyphae of the arbuscular mycorrhizal (AM) fungal genera Acaulospora, Glomus, and Scutellospora, although we found only spores of Glomus. Some roots had partial sheaths and inter- and intracellular septate fungi without indications of root necrosis. Ectomycorrhizal root tips were also found, including Cenococcum and other unknown taxa. Sporocarps of EM fungi including species of Rhizopogon, Pisolithus, Balsamia, Laccaria, Hygrophorus, and Cortinarius were found in the stand, with no other EM or arbutoid mycorrhizal plants nearby. These observations indicate that A. fasciculatum forms mycorrhizae with AM, septate, and EM fungi, but often fails to form easily recognizable mycorrhizal structures. Accepted: 5 September 1998     

Wood-decay fungi in fine living roots of conifer seedlings

The mycorrhizal basidiomycetes are known to have multiple, independent evolutionary origins from saprotrophic ancestors. To date, a number of studies have revealed functional resemblance of mycorrhizal fungi to free-living saprotrophs, but information on the ability of saprotrophic fungi to perform as mycorrhizal symbionts is scarce. Here, the objective was to investigate the ability of three wood-decay fungi, Phlebiopsis gigantea, Phlebia centrifuga and Hypholoma fasciculare, to colonize fine roots of conifer seedlings. For each fungus, mycorrhizal syntheses were attempted with Picea abies and Pinus sylvestris. After 24 wk, isolation of fungi and direct sequencing of fungal internal transcribed spacer (ITS) rDNA were carried out from healthy-looking surface-sterilized root tips that yielded both pure cultures and ITS sequences of each inoculated strain. Mycelial mantle of P. gigantea was frequently formed on root tips of P. abies, and microscopical examination has shown the presence of intercellular hyphae inside the roots. The results provide evidence of the ability of certain wood-decay fungi to colonise fine roots of tree seedlings.     

Mycorrhizae between black locust (Robinia pseudoacacia) and Terfezia terfezioides

 In the present paper we report a mycorrhizal association between the hypogeous white truffle Terfezia terfezioides and the black locust (Robinia pseudoacacia) growing at various sites in Hungary. The mycorrhiza can be considered as being of the endo- or ectendo-type, since both mantle and Hartig net are absent. Morphological features of the septate hyphae colonizing cortical root cells were investigated by light microscopy on cryosections and on ultra-thin sections studied by transmission electron microscopy. Artificial infection of micropropagated black locust plantlets with the mycelium of the fungus was successful and had the same characteristics as naturally occurring associations. Accepted: 8 March 1996     

In vitro ectomycorrhizal formation in Picea glehnii seedlings

 Twenty isolates of ectomycorrhizal fungi – 3 from Picea glehnii, 12 from other coniferous trees, and 5 from decidous trees – were tested for the ability to form mycorrhizae with P. glehnii, using an in vitro synthesis technique. Macroscopically, mycorrhizal formation was observed 3 months after inoculation, when the lateral roots began to grow. Mycelial growth was observed in all inoculated treatments, generally around and along the roots. Six months after inoculation, seedlings were harvested and the mycorrhizae were observed microscopically. Fourteen of the 20 isolates formed ectomycorrhizae with a dense sheath and a deep Hartig net; 1 formed ectendomycorrhizae with a rudimentary mantle, a well-developed Hartig net and intracellular hyphae; 3 formed pseudomycorrhizae with a mantle but without the Hartig net; and only 2 of the fungi tested, Chalciporus pipeparatus 5/92 and Lyophyllum sp. 61/92, did not form mycorrhizae at all. P. glehnii was a good host species since it had low specificity to ectomycorrhizal fungi isolated from trees other than P. glehnii. Accepted: 6 May 1996     

Structural characteristics of root–fungus associations in two mycoheterotrophic species, Allotropa virgata and Pleuricospora fimbriolata (Monotropoideae), from southwest Oregon, USA

All members of the Monotropoideae (Ericaceae), including the species, Allotropa virgata and Pleuricospora fimbriolata, are mycoheterotrophs dependent on associated symbiotic fungi and autotrophic plants for their carbon needs. Although the fungal symbionts have been identified for A. virgata and P. fimbriolata, structural details of the fungal–root interactions are lacking. The objective of this study was, therefore, to determine the structural features of these plant root–fungus associations. Root systems of these two species did not develop dense clusters of mycorrhizal roots typical of some monotropoid species, but rather, the underground system was composed of elongated rhizomes with first- and second-order mycorrhizal adventitious roots. Both species developed mantle features typical of monotropoid mycorrhizas, although for A. virgata, mantle development was intermittent along the length of each root. Hartig net hyphae were restricted to the host epidermal cell layer, and fungal pegs formed either along the tangential walls (P. fimbriolata) or radial walls (A. virgata) of epidermal cells. Plant-derived wall ingrowths were associated with each fungal peg, and these resembled transfer cells found in other systems. Although the diffuse nature of the roots of these two plants differs from some members in the Monotropoideae, the structural features place them along with other members of the Monotropoideae in the “monotropoid” category of mycorrhizas.     

Four ectomycorrhizae of Pyronemataceae (Pezizomycetes) on Chinese Pine (<Emphasis Type="Italic">Pinus tabulaeformis</Emphasis>): morpho-anatomical and molecular-phylogenetic analyses

Morphological and anatomical characters of four ectomycorrhizae with affinities to the genera Humaria, Geopora, and Trichophaea of Pyronemataceae (Pezizomycetes, Ascomycota) on Chinese Pine (Pinus tabulaeformis) are described. The ectomycorrhizae are yellowish brown to brown, and have pseudoparenchymatous outer mantle layers and partially warty emanating hyphae with thick walls and without clamps. Intrahyphal hyphae are present, and no rhizomorphs are formed. The four ectomycorrhizae are distinguishable by differences in cell shape of outer mantle layers and the presence of cystidia. Ectomycorrhizae of a possible Humaria species (Pinirhiza humarioides) lack cystidia and have irregularly inflated cells on the outer mantle layer that are connected with thin septa. The two ectomycorrhizae showing probable affinities to Geopora species (“P. daqingensis” and “P. geoporoides”) possess row-like arranged cells in the outer mantle layer and cell heaps, and differ by the presence or absence of cystidia as well as by the structure of the inner mantle layers. Ectomycorrhizae likely having been formed by a Trichophaea species (“P. trichophaeoides”) have oval to polygonal cells and no cystidia. The possible taxa affiliations were assessed by molecular-phylogenetic analyses of the internal transcribed spacer (ITS) and partial large subunit (LSU) nrDNA. Morphological and anatomical characters are discussed against the background of the LSU phylogeny.     

<Emphasis Type="Italic">Mycena</Emphasis> sp., a mycorrhizal fungus of the orchid <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

An endophytic fungus, F-23, was isolated from the roots of Dendrobium officinale Kimura et Migo, an endangered Chinese medicinal plant. The sequence of the ITS region indicated that the isolate belongs to the genus Mycena. After 4 months of inoculation, the root systems of D. officinale that were inoculated with F-23 fungus were much larger than the control’s root systems. We also observed that the hyphae of F-23 penetrated the epidermal cells within the host’s roots and spread from cell to cell. A large number of pelotons existed in the root cortical cells of D. officinale inoculated with F-23 fungus. Intracellular hyphae crossing through the host walls were also observed using SEM (scanning electron microscopy). In contrast, light microscopy and SEM showed that the transverse sections of the roots of control plants remained uncolonized. Therefore, the F-23 fungus can form mycorrhizal associations with the roots of its host plant, D. officinale, and enhance the growth of seedlings and roots. In brief, Mycena sp. was identified and shown to be a mycorrhizal fungus of the epiphytic orchid, D. officinale. This might be of potential use to the mass cultivation of D. officinale under artificial conditions.     

In situ and in vitro colonization of Cathaya argyrophylla (Pinaceae) by ectomycorrhizal fungi

Cathaya argyrophylla, a critically endangered conifer, is found to grow at four isolated areas located in subtropical mountains of China. To examine the involvement and usefulness of mycorrhizas for sustaining the population of this tree, we compared the root system, morphology, and structure of mycorrhizal roots of C. argyrophylla, which were collected from a natural stand and an artificial stand, each grown at a different location. More mycorrhizal roots were found for trees from an artificial stand. The presence of extramatrical mycelium, mantle, and Hartig net revealed that C. argyrophylla formed an ectomycorrhizal association in both sampling sites. Starch granules were found in mycorrhizal roots collected only from a natural stand. The aseptic synthesis of C. argyrophylla and Cenococcum geophilum was established for the first time in vitro. Typical ectomycorrhizas formed on seedlings on RM medium containing 0.1 g/l glucose, 5 weeks after inoculation. By light microscopy, the synthesized mycorrhizas showed a thin mantle from which emanated extramatrical hyphae and highly branched Hartig net. A simple, rapid, and convenient mycorrhiza synthesis system was developed, which facilitates further studies on ectomycorrhizal development of C. argyrophylla.     

Paxillus involutus mycorrhiza attenuate NaCl-stress responses in the salt-sensitive hybrid poplar Populus×canescens

In order to characterise the effect of ectomycorrhiza on Na⁺-responses of the salt-sensitive poplar hybrid Populus × canescens, growth and stress responses of Paxillus involutus (strain MAJ) were tested in liquid cultures in the presence of 20 to 500 mM NaCl, and the effects of mycorrhization on mineral nutrient accumulation and oxidative stress were characterised in mycorrhizal and non-mycorrhizal poplar seedlings exposed to 150 mM NaCl. Paxillus involutus was salt tolerant, showing biomass increases in media containing up to 500 mM NaCl after 4 weeks growth. Mycorrhizal mantle formation on poplar roots was not affected by 150 mM NaCl. Whole plant performance was positively affected by the fungus because total biomass was greater and leaves accumulated less Na⁺ than non-mycorrhizal plants. Energy dispersive X-ray microanalysis using transmission electron microscopy analysis of the influence of mycorrhization on the subcellular localisation of Na⁺ and Cl⁻ in roots showed that the hyphal mantle did not diminish salt accumulation in root cell walls, indicating that mycorrhization did not provide a physical barrier against excess salinity. In the absence of salt stress, mycorrhizal poplar roots contained higher Na⁺ and Cl⁻ concentrations than non-mycorrhizal poplar roots. Paxillus involutus hyphae produced H₂O₂ in the mantle but not in the Hartig net or in pure culture. Salt exposure resulted in H₂O₂ formation in cortical cells of both non-mycorrhizal and mycorrhizal poplar and stimulated peroxidase but not superoxide dismutase activities. This shows that mature ectomycorrhiza was unable to suppress salt-induced oxidative stress. Element analyses suggest that improved performance of mycorrhizal poplar under salt stress may result from diminished xylem loading of Na⁺ and increased supply with K⁺.     

Extension of the phosphorus depletion zone in VA-mycorrhizal white clover in a calcareous soil

To examine the influence of vesicular-arbuscular (VA) mycorrhizal fungi on phosphorus (P) depletion in the rhizosphere, mycorrhizal and non-mycorrhizal white clover (Trifolium repens L.) were grown for seven weeks in a sterilized calcareous soil in pots with three compartments, a central one for root growth and two outer ones for hyphae growth. Compartmentation was accomplished by a 30-μm nylon net. The root compartment received a uniform level of P (50 mg kg⁻¹ soil) in combination with low or high levels of P (50 or 150 mg kg⁻¹ soil) in the hyphal compartments. Plants were inoculated withGlomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or remained uninfected. Mycorrhizal inoculation doubled P concentration in shoot and root, and increased dry weight, especially of the shoot, irrespective of P levels. Mycorrhizal contribution accounted for 76% of total P uptake at the low P level and 79% at the high P level, and almost all of this P was delivered by the hyphae from the outer compartment. In the non-mycorrhizal plants, the depletion of NaHCO₃-extractable P (Olsen-P) extended about 1 cm into the outer compartment, but in the mycorrhizal plants a uniform P depletion zone extended up to 11.7 cm (the length of the hyphal compartment) from the root surface. In the outer compartment, the mycorrhizal hyphae length density was high (2.5–7 m cm⁻³ soil) at the various distances (0–11.7 cm) from the root surface. Uptake rate of P by mycorrhizal hyphae was in the range of 3.3–4.3×10⁻¹⁵ mol s⁻¹ cm⁻¹.     

Ectomycorrhizal symbiosis in vitro between Tricholoma matsutake and Pinus densiflora seedlings that resembles naturally occurring ‘shiro’

We established an in vitro ectomycorrhizal symbiosis between Tricholoma matsutake and Pinus densiflora. Mycorrhiza formed in a substrate of Modified Norkrans' C medium and granite-based soil had features similar to those observed previously only in naturally occurring mycorrhizal system called ‘shiro,’ and promoted the growth of plants with smaller root/shoot ratios. The in vitro formation of ‘shiro’ is essential for the development of T. matsutake system to produce mushrooms and is useful for the propagation and plantation of the mycorrhizal seedlings.