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     

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.     

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.     

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.     

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     

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     

Ectomycorrhizae of Tomentella albomarginata (Thelephoraceae) on Scots pine

 The ectomycorrhizae of Tomentella albomarginata are comprehensively described and compared to ectomycorrhizae of other Tomentella species and to ectomycorrhizae of some members of Thelephoraceae ss. Stalpers and Bankeraceae ss. Stalpers. The ectomycorrhizae of T. albomarginata are characterized by a hyphal net lying on a pseudoparenchymatous mantle surface, by tubular outgrowths of irregularly angular mantle cells, and by clamp-bearing emanating hyphae. Accepted: 15 July 1995     

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.     

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     

夏块菌与青刚栎形成外生菌根形态变化的研究

夏块菌（Tuber aestivum）是一种具有较高经济价值的菌根食用菌。对夏块菌与青刚栎（Cycloba-lanopsis glauca）在形成菌根过程中不同阶段的菌根形态变化进行了研究,结果显示：用夏块菌孢子液接种青刚栎苗后,第14天起开始形成淡乳色的外生菌根,外延菌丝刚毛状;第一至第二个月可形成黄褐色、褐色外生菌根,外延菌丝刚毛或羊毛状。外生菌根为单根,长1～4mm,直径150～250μm。菌套厚12～20μm,平坦或自菌根延伸出刚毛状菌丝,外延菌丝束黄绿色;哈替氏网菌丝直径1～1.5μm。菌根老化后变暗褐或萎缩。外延菌丝束呈黄绿色是夏块菌菌根区别于其它块菌菌根最重要的形态特征。     

Root hydraulic properties and growth of balsam poplar (Populus balsamifera) mycorrhizal with Hebeloma crustuliniforme and Wilcoxina mikolae var. mikolae

The effects of an E-strain fungus (Wilcoxina mikolae var. mikolae) and an ectomycorrhizal fungus (Hebeloma crustuliniforme) on growth and water relations of balsam poplar were examined and compared in the present study. Balsam poplar roots inoculated with W. mikolae var. mikolae (Wm) exhibited structures consistent with ectendomycorrhizal (EEM) associations, including a mantle surrounding the outside of the root and an extensive Hartig net that was located between cortical cells and extended to the vascular cylinder. Roots colonized with H. crustuliniforme (Hc) developed a mantle layer, indicative of an ectomycorrhizal (ECM) association, around the outer part of the root, but no distinct Hartig net was present. Wm-colonized balsam poplar also showed increased shoot growth, stomatal conductance (g _s), and root volumes compared with non-inoculated and Hc-inoculated plants. However, Hc-inoculated plants had higher root hydraulic conductivity (L _pr) compared with non-inoculated plants and Wm-inoculated plants. These results suggest that L _pr was not a growth-limiting factor in balsam poplar and that hyphal penetration of the root cortex in itself may have little influence on root hydraulic properties.     

REPRODUCTION AND MORPHOLOGICAL VARIATION IN SOUTHERN CALIFORNIA POPULATIONS OF THE LOWER INTERTIDAL KELP EGREGIA MENZIESII (LAMINARIALES)1

Intertidal Egregia menziesii (Turner) Aresch. populations were studied at three Southern California sites to determine temporal and spatial patterns of reproduction and morphology. The timing of sporophyll production and sporophyte recruitment was similar at all sites. Sporophyll production was much greater during winter periods of colder seawater temperatures and shorter day lengths. Sporophyte recruitment occurred from spring through midsummer, ～5 months following maximal sporophyll production. Lateral blade morphologies varied in a consistent manner, suggesting a developmental mechanism for form variation in Egregia thalli. Spatulate blades dominated shorter axes and the bases of longer axes, whereas filiform laterals became abundant toward the tips of longer axes. Filiform laterals (9.8 mg O₂·g^?1·h^?1) had higher light‐saturated net photosynthetic rates than spatulate laterals (6.8 mg O₂·g^?1·h^?1), resulting in a 12% increase in the productivity of Egregia per meter of filiform frond.     

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.     

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.     

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

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

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.     

Laccaria bicolor aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides)

The development of ectomycorrhizal associations is crucial for growth of many forest trees. However, the signals that are exchanged between the fungus and the host plant during the colonization process are still poorly understood. In this study, we have identified the relationship between expression patterns of Laccaria bicolor aquaporin LbAQP1 and the development of ectomycorrhizal structures in trembling aspen (Populus tremuloides) seedlings. The peak expression of LbAQP1 was 700‐fold higher in the hyphae within the root than in the free‐living mycelium after 24 h of direct interaction with the roots. Moreover, in LbAQP1 knock‐down strains, a non‐mycorrhizal phenotype was developed without the Hartig net and the expression of the mycorrhizal effector protein MiSSP7 quickly declined after an initial peak on day 5 of interaction of the fungal hyphae with the roots. The increase in the expression of LbAQP1 required a direct contact of the fungus with the root and it modulated the expression of MiSSP7. We have also determined that LbAQP1 facilitated NO, H₂O₂ and CO₂ transport when heterologously expressed in yeast. The report demonstrates that the L. bicolor aquaporin LbAQP1 acts as a molecular signalling channel, which is fundamental for the development of Hartig net in root tips of P. tremuloides.     

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.     

Fossil ectomycorrhizae from the Middle Eocene

Fossil ectomycorrhizae were found recently among permineralized plant remains in the middle Eocene Princeton chert of British Columbia. The ectomycorrhizae are associated with roots of Pinus and have a Hartig net that extends to the endodermis, a pseudoparenchymatous mantle, and contiguous extramatrical hyphae that are simple-septate. The mycorrhizal rootlets lack root hairs and dichotomize repeatedly to form large, coralloid clusters. Reproductive structures are absent. Based on the morphological characteristics, and the identity of the host, the closely related basidiomycete genera Rhizopogon and Suillus are suggested as comparable extant mycorrhizal fungi. These exquisitely preserved specimens represent the first unequivocal occurrence of fossil ectomycorrhizae and demonstrate that such associations were well-established at least 50 million years ago.