Cytochemical evidence of a fungal cell wall alteration during infection of Eucalyptus roots by the ectomycorrhizal fungus Cenococcum geophilum

When the ectomycorrhizal fungus Cenococcum geophilum changes from a saprophytic to a symbiotic stage, its cell wall structure becomes simplified. The external hyphal wall layer which, in the saprophytic stage, is highly reactive to the Gomori-Swift test becomes poorly reactive and can no longer be distinguished from the internal wall layer in the Hartig net hyphae. The intensely stained external wall layer was also absent from pure cultures of Cenococcum geophilum grown on a medium with a low sugar content. This cell wall alteration could be due to a decrease in the amount of melanin or of melanin plus cystine-containing proteins. This change may be necessary for increased nutrient exchange between symbionts through hyphal walls.     

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.     

Uptake and transfer of nutrients in ectomycorrhizal associations: interactions between photosynthesis and phosphate nutrition

Energy-dispersive X-ray microanalytical investigations and microautoradiographic studies were carried out to examine whether the uptake and transfer of phosphate (P) by an ectomycorrhizal fungus is affected by the carbohydrate supply of its host plant. For this purpose, non-mycorrhizal seedlings of Pinus sylvestris L. and plants inoculated with the ectomycorrhizal basidiomycete Suillus bovinus (L. ex Fr.) Kuntze were placed in the dark for 7 days in advance of a P supply. The subcellular element distribution and the uptake and distribution of (33)P was analyzed in non-mycorrhizal and mycorrhizal roots of these plants and compared with plants kept constantly under normal light conditions (control plants). The results show that placing non-mycorrhizal plants in the dark in advance of the nutrient supply led to (1) a reduction of the subcellular contents of P, S and K, but to an increase in the cytoplasmic Na content, and (2) an increase of (33)P absorption and translocation to the shoot. It can be assumed that this increased inflow of (33)P in non-mycorrhizal plants was due to P starvation after suppressed photosynthesis and reduced respiration of these plants. The suppression of photosynthesis by an ectomycorrhizal host plant and the resulting lower carbohydrate supply conditions for the ectomycorrhizal fungus led to (1) a decrease of P absorption by the mycobiont, (2) a change of the P allocation in the fungal cell compartments of an ectomycorrhizal root, and (3) a reduction of P transfer to the host plant. However, microautoradiographic studies revealed that, under these conditions, P was also absorbed by the mycorrhizal fungus and translocated via the Hartig net to the host plant. In mycorrhizal roots of plants placed in the dark in advance of the nutrient supply, the cytoplasmic P content of the Hartig net was reduced and, instead, a high number of polyphosphate granules could be detected within the hyphae. The results indicate that the exchange processes between the symbionts in a mycorrhiza are possibly linked and that P uptake and translocation by an ectomycorrhizal fungus is also regulated by the carbohydrate supply of its host plant.     

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.     

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     

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

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 geophilum on a modified Murashige-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 calli 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 ectomycorrhizal-like formation/infection between root organ/callus of Cathaya argyrophylla and the ectomycorrhizal fungus Cenococcum geophilum. This culture system is useful for further investigation of mycorrhizal synthesis in Cathaya trees.（Author for correspondence. Tel: +86 (0)451 8219 1783; Fax: +86 (0)451 8219 1795; E-mail: lumin-fu@163.com）     

The spatial distribution of acid phosphatase activity in ectomycorrhizal tissues depends on soil fertility and morphotype, and relates to host plant phosphorus uptake

Acid phosphatase (ACP) enzymes are involved in the mobilization of soil phosphorus (P) and polyphosphate accumulated in the fungal tissues of ectomycorrhizal roots, thereby influencing the amounts of P that are stored in the fungus and transferred to the host plant. This study evaluated the effects of ectomycorrhizal morphotype and soil fertility on ACP activity in the extraradical mycelium (ACP(myc)), the mantle (ACP(mantle)) and the Hartig net region (ACP(Hartig)) of ectomycorrhizal Nothofagus obliqua seedlings. ACP activity was quantified in vivo using enzyme-labelled fluorescence-97 (ELF-97) substrate, confocal laser microscopy and digital image processing routines. There was a significant effect of ectomycorrhizal morphotype on ACP(myc), ACP(mantle) and ACP(Hartig), while soil fertility had a significant effect on ACP(myc) and ACP(Hartig). The relative contribution of the mantle and the Hartig net region to the ACP activity on the ectomycorrhizal root was significantly affected by ectomycorrhizal morphotype and soil fertility. A positive correlation between ACP(Hartig) and the shoot P concentration was found, providing evidence that ACP activity at the fungus:root interface is involved in P transfer from the fungus to the host. It is concluded that the spatial distribution of ACP in ectomycorrhizas varies as a function of soil fertility and colonizing fungus.     

天麻吸收蜜环菌营养机制的细胞学研究

天麻(Gastrodia elata BI.)地下块茎皮层内具三种染菌细胞:通道细胞、寄主细胞和消化细胞。超微结构的研究表明,通道细胞被真菌所破坏,寄主细胞与真菌保持共生关系,而消化细胞能反寄生于真菌并从真菌摄取营养。消化细胞首先释放溶酶体小泡消化真菌,然后通过内吞管和内吞泡吸收菌丝细胞质降解后渗漏的可溶性有机大分子物质,后期通过消化泡进一步吞噬和消化不溶性菌丝细胞壁物质。     

A Cytological Study on the Nutrient Uptake Mechanism of a Saprophytic Orchid Gastrodia elata

There are three type cells infected by the mycorrhizal fungus, ArmiUaria mellea (Vahl ex Fr. ) Karst in the tube cortex of Gastrodia elata BI., namely the passage cells, host cells and digesting cells. Ultrastructural study demonstrated that the passage cells were distroyed by the hyphae, the host cells kept symbiotic with the hyphae, but the digesting cells could become inversely parasitic on the hyphae from which nutrient were being uptaken. The detail process of the digesting cells obtaining nutrient'from the fungus is described as follows: Firstly the digesting cells began to attack the invading hyphae by releasing numerious electron-transparent vesicles of lysosomal property, secondly they took up the soluble organic material leaked out from the digested hyphae by forming many electron-dense endocytic tubes and vesicles, and finally they endocytosed and hydrolysed the insoluble hyphal walls by forming large digesting vacuole in which a piece of hyphal wall was completely enveloped.     

赤松不定根和愈伤组织与松口蘑外生菌根菌的相互作用

本实验报道了以简单的离体培养方式来诱导赤松不定根和愈伤组织与松口蘑的菌根反应。不定根和愈伤组织均起源于无菌苗的下胚轴,接种2周后,菌丝体开始包围不定根。接种3周后,菌丝体出现在不定根皮层细胞间,哈蒂氏网型的形成也同时被确认。在愈伤组织培养物中,细胞间也能观察到菌丝体及拟-哈蒂氏网结构。这是第一个离体条件下成功地诱导赤松培养组织与松口蘑形成外生菌根的报道。     

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.     

Comparative studies on ectomycorrhizae synthesized with various in vitro techniques using Picea abies and two Hebeloma species

Summary Various in vitro synthesis techniques with Picea abies and two Hebeloma species showed that structures of the mantle and Hartig net of synthesized ectomycorrhizae within the given two fungus species are stable. However, thickness of mantle, and penetration depth and number of hyphal cell rows between cortical cells of the Hartig net are dependent on techniques and substrates. Porous glass balls as substrate in the Erlenmeyer technique seem to suppress or delay mantle and Hartig net formation. With the other techniques (growth pouch, open cuvette, Erlenmeyer with a vermiculite-peat moss mixture) development of the mantle is simultaneous with or shortly in advance of Hartig net formation. The ectomycorrhizae of the two tested Hebeloma species are similar and cannot be morphologically differentiated by the in vitro techniques used.     

Modifications of symbionts during ectomycorrhiza formation what do we know and where do we go?

Abstract

Ectomycorrhizas are subterranean organs resulting from the alteration in root structure by soil-inhabiting symbiotic fungi. Hyphae of the mycobiont have to contact the root surface, become attached to the root, and subsequently enter the root by growing between epidermal cells (and in some species, cortical cells) to form the Hartig net. A chemotropic stimulus might be involved in early hypha-root contact and recognition-adhesion may involve a polysaccharide-lectin interaction, but further research is needed to confirm this. Fungal hyphae adhering to the root surface change their mode of growth from apical, extension growth to a loss of this pattern resulting in a multi-branched mycelium. A similar change in pattern of branching occurs as hyphae form the Hartig net. In both cases, a change in the cytoskeleton might precede the change in branching. The ingress of hyphae between epidermal cells in angiosperm roots triggers radial rather than axial elongation of these cells; a reorientation of the cytoskeleton and subsequently the cellulose microfibrils is hypothesized to be involved in this process. Wall changes in root cells contiguous to Hartig net hyphae also occur, and these might facilitate nutrient exchange between the symbionts.     

Comparative structure of vesicular-arbuscular mycorrhizas and ectomycorrhizas

During the establishment of vesicular-arbuscular mycorrhizas, fungal hyphae contact the root surface, form appressoria and initiate the internal colonization phase. Structural changes occur in the cell wall, the cytoplasm and the nucleus as the fungus progresses from a presymbiotic to a symbiotic phase. Nuclei in spores are in G₁ whereas in intraradical hyphae they are in G₁ and G₂. Changes in nuclear organization are evident in various stages in the colonization process. Dramatic changes in both symbionts occur as the nutrient exchange interface is established between arbuscules and root cortical cells. An interfacial matrix, consisting of molecules common to the primary wall of the cortical cell, separates the cortical cell plasma membrane from the fungal cell wall. Ectomycorrhizas are characterized structurally by the presence of a mantle of fungal hyphae enclosing the root and usually an Hartig net of intercellular hyphae characterized by labyrinthine branching. As hyphae contact the root surface, they may respond by increasing their diameter and switching from apical growth to precocious branching. The site of initial contact of hyphae may be either the root cap or the ‘mycorrhiza infection zone’. The mantle varies considerably in structure depending on both the plant and fungus genome. In some ectomycorrhizas, the mantle may be a barrier to apoplastic transport, and in most it may store polyphosphate, glycogen, lipids and perhaps protein.     

The Multifunctional Role of Ectomycorrhizal Associations in Forest Ecosystem Processes

Belowground biological interactions that occur among plant roots, microorganisms and animals are dynamic and substantially influence ecosystem processes. Among these interactions, the ectomycorrhizal (ECM) symbiosis is remarkable but unfortunately these associations have mainly been considered within the rather narrow perspective of their effects on the uptake of dissolved mineral nutrients by individual plants. More recent research has placed emphasis on a wider, multifunctional perspective, including the effects of ectomycorrhizal symbiosis on plant and microbial communities, and on ecosystem processes. This includes mobilization of N and P from organic polymers, release of nutrients from mineral particles or rock surfaces via weathering, effects on carbon cycling, interactions with mycoheterotrophic plants, mediation of plant responses to stress factors such as drought, soil acidification, toxic metals, and plant pathogens, rehabilitation and regeneration of degraded forest ecosystems, as well as a range of possible interactions with groups of other soil microorganisms. Ectomycorrhizas are almost invariably characterized by a Hartig net composed of highly branched hyphae which entirely surround the outer root cortical cells. The Hartig net is the place of massive bidirectional exchanges of nutrients between the host and the fungus. Through these branched hyphae ectomycorrhizal fungi connect their plant hosts to the heterogeneously distributed nutrients required for their growth, enabling the flow of energy-rich compounds required for nutrient mobilization whilst simultaneously providing conduits for the translocation of mobilized products back to their hosts. In addition to increasing the nutrient absorptive surface area of their host plant root systems, the extraradical mycelium of ectomycorrhizal fungi provides a direct pathway for translocation of photosynthetically derived carbon from their hosts to microsites in the soil and a large surface area for interaction with other soil micro-organisms. The detailed functioning and regulation of these mycorrhizosphere processes is still poorly understood and needs detailed molecular approach to study these mycorrhizosphere processes but recent progress in ectomycorrhizal associations is reviewed and potential benefits of improved understanding of mycorrhizosphere interactions are discussed.     

Biosynthesis and Secretion of Indole-3-Acetic Acid and Its Morphological Effects on Tricholoma vaccinum-Spruce Ectomycorrhiza

Fungus-derived indole-3-acetic acid (IAA), which is involved in development of ectomycorrhiza, affects both partners, i.e., the tree and the fungus. The biosynthesis pathway, excretion from fungal hyphae, the induction of branching in fungal cultures, and enhanced Hartig net formation in mycorrhiza were shown. Gene expression studies, incorporation of labeled compounds into IAA, heterologous expression of a transporter, and bioinformatics were applied to study the effect of IAA on fungal morphogenesis and on ectomycorrhiza. Tricholoma vaccinum produces IAA from tryptophan via indole-3-pyruvate, with the last step of this biosynthetic pathway being catalyzed by an aldehyde dehydrogenase. The gene ald1 was found to be highly expressed in ectomycorrhiza and induced by indole-3-acetaldehyde. The export of IAA from fungal cells is supported by the multidrug and toxic extrusion (MATE) transporter Mte1 found in T. vaccinum. The addition of IAA and its precursors induced elongated cells and hyphal ramification of mycorrhizal fungi; in contrast, in saprobic fungi such as Schizophyllum commune, IAA did not induce morphogenetic changes. Mycorrhiza responded by increasing its Hartig net formation. The IAA of fungal origin acts as a diffusible signal, influencing root colonization and increasing Hartig net formation in ectomycorrhiza.     

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.     

Nitrogen delivery to maize via mycorrhizal hyphae depends on the form of N supplied

Arbuscular mycorrhizal fungi can enhance nutrient acquisition by a plant via their extraradical hyphae. This is particularly true for phosphorus, but the case for nitrogen (N) has been less clear. In our growth systems there was a small air-gap between root and hyphal compartments, which eliminated diffusion of nutrients between compartments. Moreover, our methods allowed us to distinguish between nitrate and ammonium. We found that N transfer to Zea maize L. depends on the sources fed to the hyphae of Glomus aggregatum Schenck & Smith. In experiment 1, despite the fact that plant demand for N was already met, plants received 10 times as much ¹⁵N from ammonium than from nitrate. In experiment 2, 74% of shoot-N was derived from the slow-release urea added to the hyphal compartment while only 2.9% was derived from the nitrate-N. Intraradical hyphae isolated from roots contained a considerable amount of ¹⁵N in the cell wall even when ¹⁵N-nitrate was the source. We conclude that the mycorrhizal fungus can rapidly deliver ammonium-N to the plants, and that while the fungus can absorb nitrate, it apparently lacks the capacity to transfer it to the plant.