HARTIG NET STRUCTURE OF ECTOMYCORRHIZAE SYNTHESIZED BETWEEN LACCARIA BICOLOR (TRICHOLOMATACEAE) AND TWO HOSTS: BETULA ALLEGHANIENSIS (BETULACEAE) AND PINUS RESINOSA (PINACEAE)

Hartig net structure and ontogeny were compared in ectomycorrhizae synthesized between the broad host range fungus, Laccaria bicolor and two hosts, Betula alleghaniensis and Pinus resinosa. In B. alleghaniensis, the Hartig net was present in the epidermis of the three ectomycorrhizal types formed, fast-growing first-order laterals with proximal colonization, clavate second-order laterals, and nonclavate second-order laterals. Root hair-fungus interactions occurred in this association. In P. resinosa, the Hartig net developed in epidermal and cortical cell layers of monopodial and dichotomously branched first-order laterals. Short monopodial laterals exhibited a mantle only. Fungal hyphae in the Hartig net exhibited a complex labyrinthine mode of growth in ectomycorrhizae of both tree species.     

Protein synthesis inPinus resinosa and the ectomycorrhizal fungusPaxillus involutus prior to ectomycorrhiza formation

Summary Seedlings of Pinus resinosa Ait. in test tubes were inoculated with the ectomycorrhizal fungus Paxillus involutus Fr. or with discs of sterile modified Melin-Norkrans (MMN) medium. Paxillus involutus was also inoculated to control tubes in the absence of Pinus resinosa seedlings. In vivo labelling of proteins in Pinus resinosa roots and in Paxillus involutus mycelium was carried out using ³⁵S l-methionine 1, 2, 3, 4, 5 and 7 days after inoculation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) of the protein extracts from the four treatments and autoradiography demonstrated that the presence of root exudates altered protein synthesis in Paxillus involutus as three major bands disappeared when Paxillus involutus was exposed to root exudates. Protein synthesis in Pinus resinosa was also altered when Paxillus involutus was introduced into the tubes, since at least two bands were more intense when seedlings were inoculated with Paxillus involutus, as compared to control roots. No difference was observed in the growth and the label incorporation of Paxillus involutus growing with or without root exudates. Ectomycorrhizal roots were not formed during this experiment. Gene regulation in this ectomycorrhizal association occurs, therefore, prior to the formation of ectomycorrhizal roots.     

In vitro ectomycorrhiza formation between Abies firma and Pisolithus tinctorius

 The first in vitro aseptic synthesis of Abies firma Sieb. et Zucc. with Pisolithus tinctorius (Pers.) Coker & Couch is reported. Techniques were improved for the aseptic synthesis of ectomycorrhizas of A. firma, a slow-growing species in vitro, and Pisolithus tinctorius using a novel culture medium and both sterilized and re-rooted seedlings. After 2–3 months incubation, ectomycorrhizas were formed by both methods. The mycorrhizas possessed a mantle and a highly branched nonseptate Hartig net mycelium colonizing the intercellular spaces within the host cortex, features characteristic of ectomycorrhizas. These techniques will prove useful for addressing physiological and biochemical questions on the interactions of microbes with roots of whole plants. Accepted: 29 June 1999     

The mycorrhizal fungus Paxillus involutus transports magnesium to Norway spruce seedlings. Evidence from stable isotope labeling

Although it is well established that ectomycorrhizas improve the mineral nutrition of forest trees, there has been little evidence that they mediate uptake of divalent cations such as Mg. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus Batsch in a sand culture system with two compartments separated by a 45-μm Nylon mesh. Hyphae, but not roots, can penetrate this net. Labeling the compartment only accessible to hyphae with ²⁵Mg showed that hyphae of the ectomycorrhizal fungus Paxillus involutus transported Mg to their host plant. No label was found in nonmycorrhizal control plants. Our data support the idea that ectomycorrhizas are important for the Mg nutrition of forest trees. This revised version was published online in June 2006 with corrections to the Cover Date.     

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⁺.     

Interaction between the Ectomycorrhizal Fungus Paxillus involutus, Damping-off Fungi and Pinus resinosa Seedlings

Paxillus involutus, an ectomycorrhizal fungus, had an inhibitory effect on the root pathogenic fungus Fusarium moniliforme and two isolates of F. oxysporum when grown in paired cultures on modified Melin Norkrans’ medium. In contrast, one isolate of F. oxysporum was not inhibited and another damping-off fungus, Cylindrocarpon destructans inhibited growth of Pax. involutus in similar paried cultures. Survival of Pinus resinosa (red pine) seedlings was increased significantly when they were grown in vitro concomitantly with either Pax. involutus and F. moniliforme or Pax. involutus and the three isolates of F. oxysporum, compared with seedlings inoculated with either F. moniliforme or F. oxysporum isolates alone. pax. involutus showed no protective effect against C. destructans. The number of colony forming units of Fusarium spp. was reduced significantly in the root extract and rhizosphere substrate of P. resinosa seedlings inoculated with Pax. involutus. Spore germination of Fusarium spp. was reduced significantly when treated with culture filtrate of Pax. involutus and root extract of P. resinosa seedlings inoculated with Pax. involutus. Neither colony forming units nor spore germination of C. destructans was affected either by culture filtrate of Pax. involutus or root extract of P. resinosa seedlings inoculated with Pax. involutus.     

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     

Ecological and anatomical characterization of some Pinus patula ectomycorrhizas from Mpumalanga, South Africa

 Ectomycorrhizal (ECM) fungi are an important component of the Pinus patula Schlechdt. et Cham. forest ecosystem in Mpumalanga, South Africa. ECM roots are intimately associated with accumulated litter on the forest floor and four ECM isolates where examined to determine whether they provide plant access to inorganic and organic sources of nitrogen (N) and inorganic, complexed inorganic and organic sources of phosphorus (P). In in vitro studies, all isolates were found to utilize most of the organic N compounds and organic or complexed inorganic P compounds supplied. Thus, ECM fungi could play a significant role in providing N and P to P. patula, especially from sources to which the host plant would not normally have access. Temperature sensitivities and pH optima of the four isolates differed. Of the ECM isolates WITS 01 and WITS 06 were collected from a high-litter site; WITS 01 mycorrhizas, identified as Scleroderma citrinum, were white, smooth and dichotomously branched with smooth, pale yellow, differentiated rhizomorphs. The mantle was plectenchymatous with outer and inner layers showing ring-like arrangements of hyphal bundles. The Hartig net had a palmetti shape. The WITS 02 (not identified) mycorrhizas were brown with lighter coloured root tips, with simple to dichotomous branching, smooth with no distinct mantle and sparse hyphae occurred on the root surface. The Hartig net was palmetti type with lobed haustoria. The results are discussed in relation to ECM distribution and function in nutrient cycling. Accepted: 7 January 1999     

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.     

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 structural study ofQuercus serrata andQ. acutissima formed byPisolithus tinctorius andHebeloma cylindrosporum

Ectomycorrhizas were synthesized in pots and growth pouches betweenQuercus serrata, Q. acutissima, and two ectomycorrhizal fungi,Pisolithus tinctorius andHebeloma cylindrosporum. Root morphology and the structure of the mantle and Hartig net were compared using light, fluorescence, scanning and transmission electron microscopy.P. tinctorius initially colonized root cap cells, and eventually produced a highly branched lateral root system with a complete mantle, whereasH. cylindrosporum promoted root elongation with few hyphae on the root apex surface indicating that interaction between roots differs with fungal species. Hartig net structure and hyphal inclusions varied between all the combinations tested. There were structural differences between mycorrhizas ofH. cylindrosporum/Q. acutissima grown in soil and growth pouches, which indicate that the growth pouch environment can induce artefacts in roots. Fruit bodies ofH. cylindrosporum developed in pots withQ. acutissima. AlthoughP. tinctorius has been used to inoculate oak seedlings in the nursery, results of this study indicate thatH. cylindrosporum may also be an effective ectomycorrhizal fungus forQ. serrata andQ. acutissima.     

Morphology,Anatomy and Histochemistry of Fagus grandifolia Ehrh. (North American Beech) Ectomycorrhizas

Abstract: Five distinct ectomycorrhizal morphotypes were recognized on root systems of Fagus grandifolia collected in a maple-beech woodlot. Three morphotypes showed extensive root branching whereas two had limited root branching. One morphotype, a bright orange, smooth type, was studied in detail anatomically. A compact mantle with few emanating extraradical hyphae covered the root apex of each mycorrhizal lateral. A Hartig net extended around epidermal and hypodermal cells. Roots were mostly diarch and a single-layered endodermis and a biseriate hypodermis with the inner hypodermis having suberized walls were present in all specimens examined. The mantle, and to a lesser extent the Hartig net, stored lipids, polysaccharides and proteins.     

Aseptic ectomycorrhizal synthesis betweenAbies firma andCenococcum geophilum in artificial culture

A simple in vitro system is described for the synthesis ofAbies firma-Cenococcum geophilum ectomycorrhizas. SterilizedA. firma seedlings on both MMN and FH media were inoculated with hyphal discs from actively growing margins ofC. geophilum colonies. Typical ectomycorrhizas formed on seedlings on FH medium after 3 mo of incubation. By light microscopy, the synthesized mycorrhizas were seen to possess a thin mantle from which emanated extraradicle hyphae and highly branched, rarely septate intracortical Hartig net mycelium, characteristic ectomycorrhizal features. This is the first report of aseptic ectomycorrhization ofA. firma seedlings byC. geophilum. This model system will facilitate detailed studies on ectomycorrhizal development ofAbies species.     

Paxillus involutus/Pinus sylvestris Mycorrhizae from Heavily Polluted Forest II. Ultrastructural and Cytochemical Observations

Paxillus involutus/Pinus sylvestris mycorrhizae, collected from experimental plots (situated in a Pino-Quercetum forest) treated with cadmium dust, were previously reported to accumulate metals like Cd, P, S, N, Al and Si in vacuolar bodies. The Gomori-Swift test was applied to the same material to reveal if cysteine-rich proteins might be involved in element detoxification. Vacuolar deposits of high and low phosphate level were both stained. Higher intensity of the Swift reaction was found in the fungal mycelium from mycorrhizae sampled from polluted plots than in mycorrhizae from unpolluted sites. In both cases a higher intensity of the test was revealed in the host cytoplasm close to the Hartig net than in internal parts of roots. Different intensities of the Gomori-Swift reaction were also observed when the Hartig net was compared to the fungal mantle. A PATAg test was additionally processed and revealed an accumulation of glycogen in rosettes and net-like structures when samples from cadmium dust treated plots were observed.     

Biology of the ectomycorrhizal fungal genus, Rhizopogon

The morphology and anatomy of ectomycorrhizas of Rhizopogon arctostaphyli , R. ellenae , R. flavofibrillosus , R. occidentalis , R. rubescens , R. smithii , R. subcaerulescens and R. truncatus synthesized on Ponderosa pine ( Pinus ponderosa ) in glasshouse conditions using spore slurries, are described and compared. All species produced a well-developed Hartig net, and a well-developed fungal mantle. The mantles of R. arctostaphyli , R. smithii and R. subcaerulescens ectomycorrhizas were two-layered with outer mantle hyphae of wider diameter than inner mantle hyphae. The mantle of R. subcaerulescens ectomycorrhizas also had distinctive peg-like structures (cystidia) along peripheral hyphae. Rhizopogon truncatus ectomycorrhizas were tuberculate in morphology and had a rind-like mantle enclosing adjacent roots. In addition, several species exhibited crystal inclusions in the outer mantle, presumably at the interface between mantle and soil.     

Ectomycorrhiza formation between Pseudotsuga menziesii seedling roots and monokaryotic and dikaryotic isolates of Laccaria bicolor

Seedling roots of Pseudotsuga menziesii were colonized with three monokaryotic isolates and one dikaryotic isolate of Laccaria bicolor to assess the effect of fungal genotype on ectomycorrhiza formation. Ectomycorrhizas resulting from colonization by the dikaryotic isolate had a multilayered mantle and a cortical Hartig net. One monokaryotic isolate (ss7) formed ectomycorrhizas comparable in anatomy to those induced by the dikaryotic isolate. Two other monokaryotic isolates (ss5, ss1) failed to form mantles or Hartig nets. Roots colonized by these isolates developed characteristics indicating an incompatible reaction.     

Studies in tetrapartite symbioses

Alnus incana seedlings were successfully inoculated with an endomycorrhizal fungus (Glomus fasciculatus), an ectomycorrhizal fungus (Paxillus involutus) and an isolate ofFrankia (ACN1) simultaneously. The effects of the inoculation treatments on the growth performance of the seedlings were evaluated under controlled conditions.The overall growth performance of the seedlings inoculated with the three organisms was better than those inoculated withFrankia, G. fasciculatus andP. involutus individually or withFrankia+G. fasciculatus andFrankia+P. involutus combinations. The highest growth performance and mycorrhizal infection occurred when the seedlings were inoculated simultaneously withFrankia+G. fasciculatus+P. involutus.     

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