Formation of Mycorrhiza-like Structures in Cultured Root/Callus of Cathaya argyrophylla Chun et Kuang Infected with the Ectomycorrhizal Fungus Cenococcum geophilum Fr.

An in vitro system was used for ectomycorrhizal synthesis of Cenococcum geophilum Fr. with Cathaya argyrophylla Chun et Kuang, an endangered species. Calli initiated from stem segments and adventitious roots differentiated from young seedlings were removed and cocultured with Cenococcum geophllum on a modified Murashlge-Skoog medium. Fungal hyphae were visible within intercellular spaces of the callus 4 weeks after inoculation, but definite and well-developed Hartig net structures did not form in the calU 8 weeks after inoculation. The typical ectomycorrhizal structures （i.e. hyphal mantle and Intracortical Hartig net） were observed in root segments 8 weeks after inoculation. This is the first report of aseptic ectomycorrhlzal-like formation/infection between root organ/callus of Cathaya argyrophylla and the ectomycorrhizal fungus Cenococcum geophflum. This culture system is useful for further investigation of mycorrhizal synthesis in Cathaya trees.     

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     

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     

Mycorrhiza of the host-specific Lactarius deterrimus on the roots of Picea abies and Arctostaphylos uva-ursi

The ectomycorrhizal basidiomycete species Lactarius deterrimus Gröger is considered to be a strictly host-specific mycobiont of Picea abies (L.) Karst. However, we identified arbutoid mycorrhiza formed by this fungus on the roots of Arctostaphylos uva-ursi (L.) Spreng. in a mixed stand at the alpine timberline; typical ectomycorrhiza of P. abies were found in close relation. A. uva-ursi is known as an extremely unspecific phytobiont. The mycorrhizae of both associations are described and compared morphologically. The mycorrhiza formed by L. deterrimus on both A. uva-ursi and P. abies show typical ectomycorrhizal features such as a hyphal mantle and a Hartig net. The main difference between the mycorrhizal symbioses with the different phytobionts is the occurrence of intracellular hyphae in the epidermal cells of A. uva-ursi. This emphasizes the importance of the plant partner for mycorrhizal anatomy. This is the first report of a previously considered host-specific ectomycorrhizal fungus in association with A. uva-ursi under natural conditions. The advantages of this loose specificity between the fungus and plant species is discussed.     

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.     

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 types and endobacteria associated with ectomycorrhizas of Morchella elata (Fr.) Boudier with Picea abies (L.) Karst

Subterranean morel sclerotia connected with ascomata of Morchella elata (Fr.) Boudier were found to surround 155 ectomycorrhizal root tips of Picea abies (L.) Karst, belonging to seven different types. Based upon anatomical and cytological studies, three ectomycorrhizal types could be attributed to types already described, whereas four types appeared to be undescribed. The nature of the association between the morel and the mycorrhizal types was dependent on the type and was not related to their vitality. In particular, morel ectomycorrhizas formed secondarily and exclusively by succeeding to primary mycorrhizas of a heterobasidiomycete. In addition to this triple association, an endobacterium was observed growing within the Hartig net of this heterobasidiomycete mycorrhiza. The significance of this complex of associations for the formation of ectomycorrhizas by the morel is discussed.     

A sheathing mycorrhiza between the tropical bolete Phlebopus spongiosus and Citrus maxima

Phlebopus (Ph.) spongiosus was recently described from several pomelo orchards (Citrus maxima) in southern Vietnam. This fungus was suspected to associate with pomelo plants as an ectomycorrhiza, although members of the genus Phlebopus have previously been presumed saprotrophic. To clarify this association, pomelo roots collected from the orchard (in situ roots), and those cultured with Ph. spongiosus (in vitro roots) in test tubes for 12 wk, were examined for ectomycorrhizal colonization. Both in vitro and in situ roots were analyzed for colonization using fungal LSU nuclear ribosomal DNA sequencing. The in situ roots exhibited the anatomical features of ectomycorrhizae: a thick fungal mantle, Hartig net, and extramatrical hyphae. The Hartig net, however, was very rare and showed discontinuous development. The in vitro association between Ph. spongiosus and C. maxima showed ectomycorrhiza-like structures, i.e., mantles and rhizomorphs in the plant roots, but no Hartig net development in the roots. Continuous hyphal penetration was restricted to the exodermis in both in situ and in vitro roots. Although the association between Ph. spongiosus and C. maxima could be considered ectomycorrhizal, its anatomy matches the unique feature known as sheathing mycorrhiza.     

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     

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.     

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 role of the motile tubular vacuole system in mycorrhizal fungi

Mycorrhizal fungi, to be effective for the plant, must be able to transfer mineral nutrient elements from sites of uptake at hyphal tips across various distances to the exchange region in the mycorrhiza. Vacuoles are likely to be important in this transport, since they contain elements of nutritional significance in abundance. In tip cells of hyphae of most fungi –- known to include three ectomycorrhizal basidiomycetes, an ericoid mycobiont, and two arbuscular mycorrhizal fungi –- the vacuoles form a motile tubular reticulum. The vacuoles are most active in hyphal tips, but non-motile vacuoles at a distance from the tip can be induced to become motile by environmental changes. Neither the tubular vacuolar reticulum nor its contents are properly preserved by conventional fixation and embedding. Vacuolar tubules are readily shown in vivo with fluorescent tracers, throughout the extramatrical mycelium and in outer hyphae of the sheath in eucalypt mycorrhizas synthesised with Pisolithus sp., but they have proved harder to label in field-collected ectomycorrhizas and ericoid mycorrhizas. Freeze-substitution does preserve the structure of vacuoles and vacuolar tubules, and careful anhydrous techniques allow them to be microanalysed, indicating high content of K and P in vacuoles of hyphal tips, and also in sheath and Hartig net of ectomycorrhizas. Vacuoles contain polyphosphate in diffuse, non-granular form. Polyphosphate is present right up to the tip region of hyphae as well as in sheath and Hartig net: thus important mineral nutrient elements are present at both ends of the long hyphal transport pathway. Exactly what happens in between, however, remains to be elucidated.     

Development and function of Pisolithus and Scleroderma ectomycorrhizas formed in vivo with Allocasuarina,Casuarina and Eucalyptus

The effect of inoculating seedlings of Eucalyptus grandis, Allocasuarina littoralis and Casuarina equisetifolia with two isolates of Pisolithus and two isolates of Scleroderma from under eucalypts was examined in a glasshouse trial. Ectomycorrhizas formed extensively on Eucalyptus (23–46% fine roots ectomycorrhizal) and Allocasuarina (18–51% fine roots ectomycorrhizal). On Casuarina, the fungi were either unable to colonize the rhizosphere (one isolate of Pisolithus), or sheathed roots, resembling ectomycorrhizas, formed on 1–2% of the fine roots. Colonization of roots by one isolate of Scleroderma resulted in the death of Casuarina seedlings. Inoculation with fungi increased shoot dry weight by up to a factor of 32 (Eucalyptus), 4 (Allocasuarina) and 3 (Casuarina). Ectomycorrhizas formed in associations with Eucalyptus and Allocasuarina had fully differentiated mantles and Hartig nets in which the host and fungal cells were linked by an extensive fibrillar matrix. Sheathed roots in Casuarina lacked a Hartig net, and the epidermis showed a hypersensitive reaction resulting in wall thickening and cell death. The sheaths are described as mantles since the density and arrangement of the hyphae in the sheaths was similar to that in mantles of the eucalypt ectomycorrhizas. The intercellular carbohydrate matrix was not produced in the Casuarina mantle in association with Pisolithus, hence the mantle was not cemented to the root. These structures differ from poorly compatible associations described previously for Pisolithus and Eucalyptus. The anatomical data indicate that ectomycorrhizal assessment based on surface morphological features may be misleading in ecological studies because compatible and incompatible associations may not be distinguishable.     

Mycorrhizas on nursery and field seedlings of Quercus garryana

Oak woodland regeneration and restoration requires that seedlings develop mycorrhizas, yet the need for this mutualistic association is often overlooked. In this study, we asked whether Quercus garryana seedlings in nursery beds acquire mycorrhizas without artificial inoculation or access to a mycorrhizal network of other ectomycorrhizal hosts. We also assessed the relationship between mycorrhizal infection and seedling growth in a nursery. Further, we compared the mycorrhizal assemblage of oak nursery seedlings to that of conifer seedlings in the nursery and to that of oak seedlings in nearby oak woodlands. Seedlings were excavated and the roots washed and examined microscopically. Mycorrhizas were identified by DNA sequences of the internal transcribed spacer region and by morphotype. On oak nursery seedlings, predominant mycorrhizas were species of Laccaria and Tuber with single occurrences of Entoloma and Peziza. In adjacent beds, seedlings of Pseudotsuga menziesii were mycorrhizal with Hysterangium and a different species of Laccaria; seedlings of Pinus monticola were mycorrhizal with Geneabea, Tarzetta, and Thelephora. Height of Q. garryana seedlings correlated with root biomass and mycorrhizal abundance. Total mycorrhizal abundance and abundance of Laccaria mycorrhizas significantly predicted seedling height in the nursery. Native oak seedlings from nearby Q. garryana woodlands were mycorrhizal with 13 fungal symbionts, none of which occurred on the nursery seedlings. These results demonstrate the value of mycorrhizas to the growth of oak seedlings. Although seedlings in nursery beds developed mycorrhizas without intentional inoculation, their mycorrhizas differed from and were less species rich than those on native seedlings.     

Ectomycorrhizal and arbuscular mycorrhizal colonization of two species of floodplain willows

To understand the relationships between the distribution of Chosenia arbutifolia and Salix sachalinensis and their mycorrhizal colonization, changes in the quality and types of ectomycorrhizas and arbuscular mycorrhizas of the seedlings of two species were studied at five different sites with different soil conditions in the floodplain of the Satsunai River, Hokkaido. High ectomycorrhizal and low arbuscular mycorrhizal colonization were found in roots of both plants. Ectomycorrhizal colonization of S. sachalinensis in wet sandy or muddy soil conditions was at the same level as that in dry gravelly sites. In contrast, ectomycorrhizal colonization of C. arbutifolia seedlings was lower from wet sandy sites than that from dry gravelly sites. In all study sites, the same three morphological types of ectomycorrhizas were dominant.     

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 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     

INTERACTION BETWEEN THE ECTOMYCORRHIZAL FUNGUS PISOLITHUS TINCTORIUS AND ROOT HAIRS OF PICEA MARIANA (PINACEAE)

The ectomycorrhizal fungus Pisolithus tinctorius interacts with roots of Picea mariana to form a typical mantle and Hartig net. Hyphae alter their growth pattern when in contact with susceptible root hairs in the mycorrhizal infection zone and grow acropetally, gradually covering the length of the hair to form a mantlelike structure. Initial contact with the hair may be influenced by a fibrillar material on the root hair surface. Although many root hairs become surrounded by fungal hyphae, they are not penetrated, and therefore are not entry points for this symbiotic fungus.     

马尾松与粘盖乳牛肝菌菌根共生体形成过程

对菌根共生机制的研究是对其进行应用的前提,到目前为止,绝大多数外生菌根（ectomycorrhiza,ECM）的建立过程尚不明晰,在一定程度上限制了这些ECM真菌在林业中的应用。本研究以我国南方地区主栽树种之一——马尾松Pinus massoniana和其林下优势ECM真菌——粘盖乳牛肝菌Suillus bovinus为材料,在无菌条件下研究两者菌根共生体形成过程的形态学特征。结果表明马尾松与粘盖乳牛肝菌的共生过程分为2个阶段：（1）预共生阶段,即物理接触之前,粘盖乳牛肝菌可通过释放挥发物和分泌物促进马尾松根系伸长和分枝;（2）共生阶段,又可分为3个时期。接种后第4天,粘盖乳牛肝菌菌丝体开始与马尾松根系接触并形成附着胞进入接触期;第7天菌丝开始侵入根系内部,侵入期开始;第28天菌套和哈氏网形成,即菌套和哈氏网形成期,该时期菌根化根尖开始膨大,随后继续发育至二叉分枝状菌根形成。在发育顺序方面,哈氏网与菌套同步发育,但哈氏网成形早于菌套。以上结果可对后续ECM共生机制的深入研究及马尾松高效菌根化育苗技术的开发提供参考。