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.     

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.     

Mycorrhizae ofMonotropastrum globosum growing in aFagus crenata forest

The achlorophyllousMonotropastrum globosum was found growing in aFagus crenata forest. Samples ofM. globosum and their interpenetrating root systems ofF. crenata were collected to investigate the mycorrhizal association.Monotropastrum globosum mycorrhizae showed thick sheaths, invasion of the epidermal cells by fungal pegs, and Hartig nets, which reached only the first layer of cortical cells. TheF. crenata mycorrhizae also showed thick sheaths, but Hartig nets penetrated deep into the cortex and intracellular hypha were seen in the outer cortical cells. The similarities observerd in the mantle inner plan view and emanating hypha suggest that both mycorrhizae are formed by the same fungus.     

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.     

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.     

Identification of Picea-ectomycorrhizae by comparing DNA-sequences

Identification of the fungi forming ectomycorrhiza is still a great challenge. Ectomycorrhizae of Picea abies, collected in southwest Germany during several years and described as morphotypes, were identified using LSU and ITS sequences. To this the fungal sequences amplified from the mycorrhizae were compared with sequences from identified sporocarps. The fungal partner of Piceirhiza gelatinosa was identified as Hygrophorus olivaceoalbus, the fungal partner of Piceirhiza rosa-nigrescens was identified as Dermocybe cf. semisanguinea, and the fungal partner of a white mycorrhiza, described here for the first time, belongs to the Hebeloma velutipes group (Hebeloma crustuliniforme complex). Identification to genus level was possible for Piceirhiza lanuginosa where a Cortinarius-species is the fungal partner. A Tomentella-species forms a newly described light-brown mycorrhiza. Identification to family and to order-level was achieved for a milky-dull silvery mycorrhiza (Cortinariaceae), and Piceirhiza globulifera (Aphyllophorales), respectively. Ten samples of white, slightly bent mycorrhizae were formed by 8 different Cortinarius-species, including among others Cortinarius traganus, C. delibutus, and C. brunneus. The fungal partners of several brown, smooth mycorrhizae displaying only a Hartig net are Ascomycetes, among these are Wilcoxina cf. mikolae and Hymenoscyphus cf. ericae.     

Apoplastic Transport through the Fungal Sheath of Pinus sylvestris/Suillus bovinus Ectomycorrhizae

Roots of Pinus sylvestris L. were inoculated in vitro with the basidiomycete Suillus bovinus (Fr.) O. Kuntze. To investigate apoplastic transport in mycorrhizal and sterile roots of Pinus sylvestris, roots of intact plants were submerged for 20 h in 0.1% solutions of the fluorescent dye sulphorhodamine G (SR-G) or for 6 h in 1.5% solutions of lanthanum nitrate. Samples treated with the dye were cryofixed, freeze-dried or freeze-substituted and embedded into Spurr's medium, maintaining strictly anhydrous conditions to prevent movement of the water-soluble dye after cryofixation. Lanthanum-treated roots were fixed in glutaraldehyde, post-fixed in OsO₄, dehydrated in a graded acetone series and embedded in Spurr's resin. The apoplastic distribution of the two tracers were examined either using fluorescence optics (sulphorhodamine) or with the electron microscope (La³⁺). The yellow-green fluorescence of sulphorhodamine could be detected within the apoplast of the fungal sheath, the Hartig net and the host cortex, up to the endodermis. Electron-dense lanthanum deposits were located in the fungal sheath, the Hartig net and in the root cortex. Greater deposition was detected within the matrix material, in which the hyphae of the mantle are embedded. The apoplastic distribution of the two tracers within the plant root did not indicate any significant qualitative differences between sterile and mycorrhizal rootlets. In contrast to reports by other authors, we conclude that the fungal sheath does provide an apoplastic pathway for water and ions at least in Pinus sylvestris/Suillus bovinus mycorrhizae. However, the mobility of charged molecules, particularly cations, may be limited by the fungal matrix.     

The mycorrhizal fungus<Emphasis Type="Italic"> Tricholoma matsutake</Emphasis> stimulates<Emphasis Type="Italic"> Pinus densiflora</Emphasis> seedling growth in vitro

While it has been suggested that Matsutake mycorrhizae might not be functional and that Matsutake may behave as a saprobic fungus in soil or even have some pathogenic activity on seedlings, we investigated the consequences of Matsutake inoculation on Pinus densiflora growth. Seventy-five days after inoculation, hyphae were anchored on short roots and well-developed Hartig net palmettis were observed. Compared to both control treatments—seedlings treated with distilled water and seedlings treated with autoclaved mycelium—inoculation significantly stimulated seedling total dry weight by 70.9% and 98.0%, respectively. These findings attest that some type of symbiotic relationship must be functional and favour host growth, ruling out claims of pathogenicity under the sterile conditions used here.     

Mycorrhiza-like interaction by Morchella with species of the Pinaceae in pure culture synthesis

 Isolates from two species of Morchella were tested for ability to form mycorrhizae in pure culture synthesis with Arbutus menziesii, Larix occidentalis, Pinus contorta, Pinus ponderosa, andPseudotsuga menziesii. Ectomycorrhizal structures (mantle and Hartig net) formed with the four species of the Pinaceae but not with A. menziesii. Results are compared to previous studies on morel mycorrhizae and discussed in an ecological context. Accepted: 23 October 1999     

Mycorrhizal features and fungal partners of four mycoheterotrophic Monotropoideae (Ericaceae) species from Yunnan, China

We provide a preliminary report of the mycobionts found within four Monotropoideae (Ericaceae) species from China: Monotropa uniflora, Hypopitys monotropa, Monotropastrum humile and Monotropastrum sciaphilum (a rare endemic species never previously studied for mycorrhizae). Such achlorophyllous Monotropoideae plants obtain their carbohydrates from mycorrhizal fungi linking them to surrounding trees, on which these fungi form ectomycorrhizae. Since Monotropoideae were rarely studied in continental Asia, the root systems of the four species sampled in Yunnan were examined using morphological and molecular methods. All the roots of these four species exhibit a typical monotropoid mycorrhizal morphology, including a fungal mantle, a Hartig net and hyphal pegs. In M. uniflora and M. humile mycorrhizae, cystidia typical of Russula symbionts covered the fungal mantle. ITS barcoding revealed that Russulales were the most frequent colonizers in all species, but Hypopitys monotropa displayed various additional mycorrhizal taxa. Moreover, a few additional ectomycorrhizal and saprotrophic Basidiomycota taxa were identified in the three other species, challenging that these four Monotropoideae species are as strictly fungal specific as the other Monotropoideae species hitherto studied. Moreover, a comparison with accompanying fungus sporocarps revealed that the fruiting fungal community significantly differed from that associated with the Monotropoideae roots, so that a clear fungal preference was evident. Finally, four fungal species were found on more than one Monotropoideae species: this contrasted with previous reports of sympatrically growing mycoheterotrophic plants, which did not reveal any overlap. This again challenges the idea of strict fungal specificity.     

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.     

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.     

Immunogold localization of glutamine synthetase and NADP-glutamate dehydrogenase of Laccaria laccata in Douglas fir ectomycorrhizas

Using antibodies raised against glutamine synthetase (GS) and NADP-glutamate dehydrogenase (NADP-GDH) from Laccaria laccata, we examined tissular localization of GS and NADP-GDH in symbiotic tissues of Douglas fir/L. laccata ectomycorrhizas by immunogold labeling. Thin sections of mycorrhizal roots were first treated either with an anti-GS- or antiNADP-GDH-specific antibody and then with a colloidal gold marker. Both enzymes appeared to be cytoplasmic. Our results also indicated the presence of GS in some fungal cells in dense cytoplasmic patches. It also appeared that GS is more abundant than NADP-GDH. The distribution of these nitrogen-assimilating enzymes in the fungal Hartig net and the sheath did not differ significantly. No labelling was observed in host cells.     

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     

Localization of chitinolytic activities in Fagus sylvatica mycorrhizas

 Localization of chitinolytic activities in Fagus sylvatica (beech) mycorrhizas was examined using a range of fluorogenic 4-methylumbelliferyl [4-MU-(GlcNAc)_1–4] substrates in order to distinguish between exochitinase, endochitinase and β-N–acetylglucosaminidase activities. The validity of the technique was confirmed using onion epidermis cells. In the beech mycorrhiza, endochitinase activity was not detectable above background fluorescence. Exochitinase activity was detected in the fungal sheath and the Hartig net. β-N–Acetylglucosaminidase activity was also mainly associated with the fungal sheath and Hartig net. Individual fungal hyphae extending from these structures also showed substantial β-N–acetylglucosaminidase activity. The cortical cell walls of the host in the Hartig net region also fluoresced brightly. The localization of β-N–acetylglucosaminidase activity was confirmed using a chromogenic histochemical reagent, 5-bromo-4-chloro-3-indolyl-N–acetyl-β-d-glucosaminide (X-GlcNAc). Accepted: 5 December 1995     

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.     

Mycorrhizal synthesis between <Emphasis Type="Italic">Lactarius deliciosus</Emphasis> and <Emphasis Type="Italic">Arbutus unedo</Emphasis> L.

Arbutoid mycorrhizae were synthesized in vitro between Arbutus unedo L. and two isolates of Lactarius deliciosus. The fungal isolates were obtained from sporocarps collected under Pinus sylvestris and in a mixed forest stand of Quercus suber and Pinus pinea. Synthesis tubes filled with a mixture of sterilized peat, vermiculite, and perlite imbibed with nutrient solution were used. Two inoculation methods using solid and liquid media were tested. Shoots from an adult selected clone of A. unedo were used after in vitro rooting by auxin dipping. After 3 months of shoots transfer to the substrate, the root systems were examined for arbutoid mycorrhizae formation and later on ex vitro conditions, 9 months after acclimatization. The inoculum treatment with liquid medium improved the mycorrhizal development for both isolates, in vitro. Sterilized substrate for plant acclimatization increased the mycorrhizal development. The arbutoid mycorrhizae were observed in vitro as well as 9 months after acclimatization. Standard arbutoid mycorrhiza features were observed: pale yellow mantle, typical cruciform appearance, Hartig net (HN), and intracellular hyphal complexes, both confined to the epidermis. L. deliciosus mycorrhizae synthetized in vitro persisted 9 months after plant acclimatization. Morphological observations were confirmed by molecular techniques.     

Relationships between Stand Composition and Ectomycorrhizal Community Structure in Boreal Mixed-Wood Forests

We investigated the community structure of ectomycorrhizal fungi under varying overstory tree compositions in the southern mixed-wood boreal forest of Quebec. Sampling took place at two locations of differing postfire ages and nine 100-m² plots were sampled per location. The dominant overstory tree species in the plots were trembling aspen (Populus tremuloides Michx.), white birch (Betula papyrifera Marsh.) or white spruce [Picea glauca (Moench) Voss], and balsam fir [Abies balsamea (L.) Mill.]. Mycorrhizae were analyzed using morphological as well as molecular methods, employing fungal-specific primers to amplify ribosomal DNA for subsequent cloning and sequencing. A total of 1800 mycorrhizal root tips collected from the 18 plots were morphologically classified into 26 morphotypes, with Cenococcum geophilum dominating (36% of root tips). A second set of root tips, selected from the same 18 samples on which the morphological analysis was based, were analyzed using molecular methods. From this analysis, 576 cloned polymerase chain reaction products were screened by restriction fragment length polymorphism analysis and a total of 207 unique types were found. No one type dominated the system and 159 occurred only once. Sequence analysis of the types that occurred more than once revealed that Piloderma sp., Russula sp., Cortinarius sp., and Lactarius sp. were the most common mycorrhizae. The ectomycorrhizal fungal community structure revealed by the rDNA analysis differed from that observed using morphological methods. Canonical correspondence analyses of the sequenced restriction types and % overstory composition indicate that the distributions of ectomycorrhizal fungi are influenced by the relative proportions of host tree species. The distinct fungal assemblages found in the different plots supported by the different combinations of host tree species provides further support for the need to conserve stand diversity in the southern boreal forest.