Structural aspects of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] tuberculate ectomycorrhizae

Summary Tubercles of Pseudotsuga menziesii consisted of clusters of ectomycorrhizae surrounded by a peridiumlike rind. Energy dispersive spectroscopy demonstrated that crystals found in the zone of loose hyphae extending from the inner rind to the mantle of each root probably contain calcium oxalate. Inner mantle and Hartig net hyphae showed a labyrinthine branching pattern and stored carbohydrates and protein. The Hartig net formed up to inner cortical cells which had thickened, darkly stained walls. Bacteria were located either along with hyphae within the rind or as colonies on the surface of the tubercle.     

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.     

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.     

Ectomycorrhizal syntheses with Picea abies and three fungal species: a case study on the use of an in vitro technique to identify naturally occurring ectomycorrhizae

Ectomycorrhizal syntheses between Picea abies and the fungal associates Scleroderma citrinum, Boletus luridus, and Tricholoma vaccinum were carried out using Melin's Erlenmeyer flask technique. The symbioses of S. citrinum were characterized by a mantle composed of an outer prosenchymatous and an inner synenchymatous layer. The mantles of B. luridus and T. vaccinum were solely prosenchymatous. Rhizomorphs were produced in all treatments, but only in association with S. citrinum were they differentiated with additional, enlarged hyphae. All synthesized ectomycorrhizae were white or whitish to light orange and greyishorange. On large-scale root sampling in two differing Picea abies forests in Switzerland, nine out of a total of 22 morphological types of ectomycorrhizae were white or yellow in colour and were, therefore, comparable with the synthesized ectomycorrhizae. These nine natural types generally had distinct mantle features (irregular synenchyma, gelatinous matrix, cystides, thick-walled hyphae), but mostly lacked clamp connections. Synthesized ectomycorrhizae, on the other hand, lacked distinct mantle characteristics and always had clamp connections. Natural and synthesized white or yellow ectomycorrhizae did not coincide morphologically and thus identification of the fungal partners of natural symbioses by means of in vitro-synthesis with potential ectomycorrhizal fungi was not possible in the present study.     

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular fungal pegs within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.     

Structure and histochemistry of mycorrhizae synthesized between Arbutus menziesii (Ericaceae) and two basidiomycetes,Pisolithus tinctorius (Pisolithaceae) and Piloderma bicolor (Corticiaceae)

Arbutoid mycorrhizae were synthesized in growth pouches between Arbutus menziesii Pursch. (Pacific madrone) and two broad host range basidiomycete fungi, Pisolithus tinctorius (Pers.) Coker and Couch and Piloderma bicolor (Peck) Jülich. P. tinctorius induced the formation of dense, pinnate mycorrhizal root clusters enveloped by a thick fungal mantle. P. bicolor mycorrhizae were usually unbranched, and had a thin or non-existent mantle. Both associations had the well-developed para-epidermal Hartig nets and intracellular penetration of host epidermal cells by hyphae typical of arbutoid interactions. A. menziesii roots developed a suberized exodermis which acted as a barrier to cortical cell penetration by the fungi. Ultrastructurally, the suberin appeared non-lamellar, but this may have been due to the imbedding resin. Histochemical analyses indicated that phenolic substances present in epidermal cells may be an important factor in mycorrhiza establishment. Analyses with X-ray energy dispersive spectroscopy showed that some of the granular inclusions present in fungal hyphae of the mantle and Hartig net were polyphosphate. Other inclusions were either protein or polysaccharides.     

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.     

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     

Comparative investigations on the differentiation of the endodermis and the development of the Hartig net in mycorrhizae of Picea abies and Larix decidua

Summary The differentiation of the endodermis of mycorrhizal roots of Picea abies and Larix decidua was investigated by means of light and transmission electron microscopy and with fluorescence techniques. The initiation and differentiation of the Hartig net were recorded. Differences between the two tree species were found, as were differences between the two tree species and angiosperms. The Casparian band developed immediately after the origin of endodermal cells from the meristem in mycorrhizae of both tree species. In L. decidua only the primary endodermis was present in most mycorrhizal laterals. The secondary structure of the endodermis was restricted to main roots and proximal parts of larch mycorrhizae. In P. abies mycorrhizae, however, the secondary stage of the endodermis developed soon after the primary endodermis and was characterized by regular alternation of short, active passage cells and elongated, rapidly degenerating cells, the inner surface of which was covered by a thick suberin layer. Hartig net development started in P. abies short roots only after the differentiation of endodermis into the secondary stage, whereas in L. decidua, the Hartig net was already initiated at the primary endodermal stage. Differences were specific for tree species.     

Ectomycorrhizae formed in vitro by quaking aspen: including Inocybe lacera and Amanita pantherina

Ectomycorrhizae formed in synthesis tubes by aspen (Populus tremuloides) seedlings and each of seven fungal isolates are described. Isolates of Amanita muscaria v. formosa, A. pantherina, Inocybe lacera, and Paxillus vernalis, from sporocarps collected in aspen stands in southwestern Montana, developed mantles and Hartig nets on aspen roots, as did the broad-hostrange fungi Cenococcum geophilum and Pisolithus tinctorius from the VPI culture collection. Chalciporus piperatus failed to form mycorrhizae, and Piloderma croceum formed a mantle, but no Hartig net. The first syntheses of I. lacera and A. pantherina with aspen are reported.     

Comparative studies on the mycorrhization of Larix decidua and Picea abies by Suillus grevillei

Summary Mycorrhization of Picea abies has been achieved, for the first time, with six strains of Suillus grevillei by a new culture method, using activated charcoal paper and liquid medium as a substrate. Mycorrhization of P. abies and Larix decidua was compared, and the process was found to be significantly different in the two tree species. S. grevillei is not incompatible with P. abies, but it forms mycorrhizae more readily with L. decidua. Hyphal growth was clearly stimulated on the surface of roots of Larix but retarded on Picea. A well organized Hartig net was formed with both tree species, but wall protuberances were frequently observed on the outer cell walls of Picea cortex cells when the Hartig net was not fully developed. No conspicuous cell wall reactions occurred in Larix roots. Cell wall protuberances may be comparable to those in transfer cells and are interpreted as an alternative to Hartig net development. Anatomical differences between roots of Larix and Picea, and physiologically active substances such as recognition factors on the root surfaces, are discussed with respect to their responsibility for the different reactions of S. grevillei.     

The influence of culture conditions on mycorrhiza formation between the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings

Interactions between an isolate of the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings were studied at the structural and ultrastructural levels. Several different conditions were tested with or without sugar and in a sterile or nonsterile medium. In the growth cabinet, the A. africana/Pisolithus sp. interactions did not produce ectomycorrhizas. A fungal sheath was formed but no Hartig net, and an unusual host epidermal cell wall was observed. Hyphae of Pisolithus sp. induced modifications of epidermal cells of 15-day-old A. africana seedlings indicative of non-mycorrhizal interactions, such as wall thickening, wall ingrowth, papillae formation, degraded host wall material and the presence of intracellular hyphae. Wall ingrowth consisted of depositions of host cell wall materials giving a positive reaction for polysaccharides; however, wall thickenings and papillae showed no homogeneous reactions for polysaccharides. In glasshouse conditions, inocula of Pisolithus sp. in the form of spores or mycelia entrapped in peat-vermiculite added to sterilized soil produced typical ectomycorrhizae only with 6-month-old A. africana seedlings. Under these conditions, no conspicuous cell wall reactions occurred on A. africana roots. The results demonstrate that the establishment of an association between an ectomycorrhizal fungus and a potential host plant is strongly influenced by seedling age and/or environmental conditions. Therefore, in vitro synthesis is not a conclusive demonstration of a symbiotic relationship.     

Effect of time of inoculation on in vitro ectomycorrhizal colonization and nodule initiation in Acacia holosericea seedlings

The complex interactions that occur in systems with more than one type of symbiosis were studied using one isolate of Bradyrhizobium sp. and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch inoculated on to the roots of Acacia holosericea A. Cunn. ex G. Don in vitro. After a single inoculation with Bradyrhizobium sp., bacteria typically entered the roots by forming infection threads in the root hair cells via the curling point of the root hair and/ or after intercellular penetration. Sheath formation and intercellular penetration were observed on Acacia roots after a single inoculation with Pisolithus tinctorius but no radial elongation of epidermal cells. Simultaneous inoculation with both microorganisms resulted in nodules and ectomycorrhiza on the root system, occasionally on the same lateral root. On lateral roots bearing nodules and ectomycorrhiza, the nodulation site was characterized by the presence of a nodule meristem and the absence of an infection thread; sheath formation and Hartig net development occurred regularly in the region of the roots adjacent to nodules. Prior inoculation with Bradyrhizobium sp. did not inhibit ectomycorrhizal colonization in root segments adjacent to nodules in which nodule meristems and infection threads were clearly present. Conversely, in ectomycorrhizae inoculated by bacteria, the nodule meristem and the infection thread were typically absent. These results show that simultaneous inoculation with both microorganisms inhibits infection thread development, thus conferring an advantage on fungal hyphae in the competition for infection sites. This suggests that fungal hyphae can modify directly and/or indirectly the recognition factors leading to nodule meristem initiation and infection thread development.     

Synthesis and establishment of <Emphasis Type="Italic">Tuber melanosporum</Emphasis> Vitt. ectomycorrhizae on two <Emphasis Type="Italic">Nothofagus</Emphasis> species in Chile

Axenically germinated seedlings of two species of Southern beech (Nothofagus obliqua, N. glauca) from Chile were inoculated with spores of the Périgord black truffle (Tuber melanosporum). Ectomycorrhizal development was monitored for 6 months in the greenhouse and compared to the performance of the natural host species Quercus ilex and Quercus robur. Seedling survival and mycorrhization showed major differences in both Nothofagus species: T. melanosporum readily formed ectomycorrhizae with seedlings of N. obliqua, although at a lower rate than with Q. ilex but at a proportion very similar to Q. robur; survival and colonization rates were high, and seedling growth was not visibly affected by the high soil pH required by T. melanosporum. In contrast, more than 50% of N. glauca seedlings died after inoculation, and mycorrhiza formation was very sparse. In both species, no colonization by adventive ectomycorrhizal fungi could be observed, whereas both species of Quercus showed minor colonization by another fungus, probably Inocybe or Hebeloma. Our results show that it is possible to infect N. obliqua with the Périgord black truffle under greenhouse conditions, which opens up the possibility of cultivating this truffle as a secondary crop during reforestation with N. obliqua in Chile.     

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.     

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     

Extracellular complexation of Cd in the Hartig net and cytosolic Zn sequestration in the fungal mantle of Picea abies – Hebeloma crustuliniforme ectomycorrhizas

Compartmentation of heavy metals on or within mycorrhizal fungi may serve as a protective function for the roots of forest trees growing in soils containing elevated concentrations of metals such as Cd and Zn. In this paper we present the first quantitative measurements by X‐ray microanalysis of heavy metals in high‐pressure frozen and cryosectioned ectomycorrhizal fungal hyphae. We used this technique to analyse the main sites of Cd and Zn in fungal cells of mantle and Hartig net hyphae and in cortical root cells of symbiotic Picea abies – Hebeloma crustuliniforme associations to gain new insights into the mechanisms of detoxification of these two metals in Norway spruce seedlings. The mycorrhizal seedlings were exposed in growth pouches to either 1 mM Cd or 2 mM Zn for 5 weeks. The microanalytical data revealed that two distinct Cd‐ and Zn‐binding mechanisms are involved in cellular compartmentation of Cd and Zn in the mycobiont. Whereas extracellular complexation of Cd occurred predominantly in the Hartig net hyphae, both extracellular complexation and cytosolic sequestration of Zn occurred in the fungal tissue. The vacuoles were presumed not to be a significant pool for Cd and Zn storage. Cadmium was almost exclusively localized in the cell walls of the Hartig net (up to 161 mmol kg ^{? 1} DW) compared with significantly lower concentrations in the cell walls of mantle hyphae (22 mmol kg ^{? 1} DW) and in the cell walls of cortical cells (15 mmol kg ^{? 1} DW). This suggests that the apoplast of the Hartig net is a primary accumulation site for Cd. Zinc accumulated mainly in the cell walls of the mantle hyphae (111 mmol kg ^{? 1} DW), the Hartig net hyphae (130 mmol kg ^{? 1} DW) and the cortical cells (152 mmol kg ^{? 1} DW). In addition, Zn occurred in high concentrations in the cytoplasm of the fungal mantle hyphae (up to 164 mmol kg ^{? 1} DW) suggesting that both the cell walls and the cytoplasm of fungal tissue are the main accumulation sites for Zn in P. abies resulting in decreased Zn transfer from the fungus to the root.     

Regulation of photoassimilate allocation in Pinus sylvestris seedlings by the nutritional status of the mycorrhizal fungus Suillus variegatus

Summary Seedlings of Pinus sylvestris L. were grown on defined nutrient solutions on carbon filters, either sterile or infected with the basidiomycete Suillus variegatus O. Kuntze. After mycorrhizas were established, the shoot of the seedling was subjected to ¹⁴CO₂ photosynthesis. ¹⁴C-labelled photoassimilates were translocated to both mycorrhizas and non-infected root tips. Microautoradiographs of mycorrhizas indicated that omission of external sugars did not affect the formation of mycorrhizas; ¹⁴C-photoassimilates were supplied to cortex, Hartig net and the mantle of hyphae surrounding the rootlet. Nutrient solution containing sugars (malt extract, glucose) enhanced the growth of the fungus. As a consequence, ¹⁴C-photoassimilates from the seedling were accumulated in the mantle, but defence mechanisms of the host cannot be excluded. When soluble nitrogen was omitted from the nutrient solution and replaced by chitin precipitated on the filter-bearing mycorrhizas, the fungus appeared strongly labelled in the mantle, where the fungal chitinase provided soluble nitrogen compounds, necessary for the growth of the seedling.     

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.     

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