Hartig Net Hyphae Research Articles

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.

Identification of cytoskeletal components in pine ectomycorrhizas

SUMMARYEctomycorrhizal associations were synthesized between pine seedlings and the fungi Suillus bovinus (L.) ex Fr. or Paxillus involutus (Batsch ex Fr.) Fr. Immunoblotting of polypeptides separated electrophoretically from crude tissue extracts revealed the abundant presence of tubulin and actin in ectomycorrhiza and lower amounts in the fungal strands surrounding the ectomycorrhizal roots. In ectomycorrhiza the α‐tubulins from fungal hyphae and plant cells were clearly distinguishable but such discrimination was not possible for β‐tubulin or actin due to the similar mobility of proteins originating from the conifer and fungal tissues. Young ectomycorrhizal short roots were fixed while still attached to the seedlings and, using indirect immunofluorescence microscopy with tubulin antibodies, microtubules were detected in both the conifer cells and in fungal hyphae. In the host plant cytoplasmic and spindle microtubules were visualized in meristem cells and in differentiating vascular tissue but not in the cortical cells. In symbiotic hyphae the microtubule tracks and spindles of dividing nuclei were clearly distinguished in the mantle hyphae in the tip region of the short roots. In the Hartig net hyphae microtubule tracks changed to a less clear, reticulate structure. Actin was visualized as long filaments in vascular tissue cells and as small microfilament bundles in mantle hyphae. Short microtubules and actin dots were detected in cytoplasm‐containing hyphae on the strand surface. The possible role of the cytoskeletal elements in the maintenance of the ectomycorrhizal association is discussed.

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

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.

Structure and development of Pinus banksiana – Wilcoxina ectendomycorrhizae

Seedlings of Pinus banksiana were grown in growth pouches and inoculated with Wilcoxina mikolae var. mikolae, Wilcoxina mikolae var. tetraspora, and Wilcoxina rehmii. Ectendomycorrhizae formed between P. banksiana and W. mikolae var. mikolae developed rapidly following inoculation. The mantle was of variable width, and a large amount of mucigel was evident on the root surface. Intracellular penetration of the cortical cells by hyphae occurred one to two cells distal to Hartig net formation. Both light and transmission electron microscopy revealed labyrinthic growth of Hartig net hyphae that were densely cytoplasmic during early penetration stages but became vacuolate as the association aged. Intracellular colonization of the cortex was extensive, with the hyphae highly branched and surrounded by an interfacial matrix and cortical cell plasma membrane. The external morphology and anatomy of ectendomycorrhizae formed between W. mikolae var. tetraspora and W. rehmii and P. banksiana were similar to those described for W. mikolae var. mikolae. Key words: ectendomycorrhizae, Wilcoxina, Pinus banksiana, intracellular, Hartig net, E-strain.

Structure and ontogeny of Betula alleghaniensis – Pisolithus tinctorius ectomycorrhizae

The ontogeny and ultrastructure of ectomycorrhizae synthesized between Betula alleghaniensis (yellow birch) and Pisolithus tinctorius, a broad host range fungus, were studied to determine the structural modifications in both symbionts during ectomycorrhiza establishment. A number of stages, including initial contact of hyphae with the root surface, early mantle formation, and mature mantle formation, were distinguished. Interactions between hyphae and root hairs were frequent. As a paraepidermal Hartig net developed, root epidermal cells elongated in a radial direction, but wall ingrowths were not formed. Repeated branching of Hartig net hyphae resulted in extensive fine branches and the compartmentalization of hyphal cytoplasm. Nuclei and elongated mitochondria were frequently located in the narrow cytoplasmic compartments, and [Formula: see text] thickenings developed along walls of cortical cells in primary roots.

Ontogeny of Alnus rubra – Alpova diplophloeus ectomycorrhizae. II. Transmission electron microscopy

Ultrastructural features of the two symbionts in ectomycorrhizae formed between Alnus rubra and Alpova diplophloeus change with developmental stage. In the root cap – meristem zone, hyphae penetrate between vacuolated root cap cells and become appressed to epidermal cells containing small vacuoles, plastids with starch, numerous Golgi bodies, mitochondria, and endoplasmic reticulum cisternae. In the young Hartig net zone, hyphae with few vacuoles penetrate between vacuolated epidermal cells that still contain numerous Golgi bodies but now have plastids with small starch grains. Hartig net hyphae begin to branch and eventually form a complex branching system in the mature Hartig net zone. Hartig net hyphae in the basal portion of the ectomycorrhizae synthesize lipid and finally become vacuolated.

Ontogeny of Alnus rubra – Alpova diplophloeus ectomycorrhizae. I. Light microscopy and scanning electron microscopy

Ectomycorrhizae synthesized between Alpova diplophloeus and Alnus rubra are of two morphological types: one with a mantle formed along the entire length of the lateral roots and the other, the clavate type, with the mantle confined to the apical portion of the laterals. The morphology of the mycorrhiza is dependent on the stage of lateral root elongation at the time of colonization by fungal hyphae. Clavate mycorrhizae form on lateral roots that have already elongated at the time of fungal colonization. Fungal hyphae interact with root hairs at the base of clavate mycorrhizae. Mantles of both types are fairly compact with few extramatrical hyphae. Hartig net hyphae, which branch profusely primarily in the radial direction, are confined to the epidermis and midway along the radial walls of the outer layer of cortical cells. Second-order lateral root primordia are initiated in the mature Hartig net zone. Cells in the outer layer of the cortex of mycorrhizal roots collapse during fixation, indicating the possible presence of a barrier in the cell wall blocking the ingress of fixative.

An Ultrastructural Study of Modifications in Dryas intergrifolia and Hebeloma cylindrosporum During Ectomycorrhiza Formation

Ectomycorrhiza formation between Dryas integrifolia and Hebeloma cylindrosporum is initiated by changes in the growth pattern of hyphae when they first contact root epidermal cells in the mycorrhizal infection zone. Fungal hyphae then grow within the middle lamella of the plant root. Fungal growth is modified and branching patterns similar to those found in the Hartig net of most ectomycorrhizal systems result. Inter-cellular hyphal growth is impeded by the deposition of wall material around plasmodesmata, the latter maintaining symplastic continuity between root cells. The middle lamella in these regions is, however, eventually occupied by growing hyphae that disrupt plasmodesmata. Transfer cell-like wall ingrowths occur in cortical cells in the Hartig net region. Ultrastructural changes occur in Hartig net hyphae and contiguous root cells during the ontogeny of the ectomycorrhiza.

Ultrastructural localization of ATPase activity in the Pinus sylvestris/Laccaria laccata ectomycorrhizal association.

Ultrastructural localization of the plasmalemmal ATPase activity has been studied in basidiomycete ectomycorrhizas synthesized between Pinus sylvestris L, and Laccaria laccata Scop, ex Fries. The cortical cells showed a strong plasmalemma-bound ATPase activity. The activity was also associated with the fungal plasmalemma in the external hyphae, sheath and Hartig net. The plasmalemmal ATPase activity disappeared in degenerating cortical cells. In the Hartig net, only the parts of the plasmalemma adjacent to other hyphae were active. When the Hartig net hyphae adjoined on one side a living cortical cell and, on the other side, a dead cortical cell, the ATPase activity of fungus plasmalemma was asymmetrically distributed. These observations are discussed in relation to the possible function of ATPase in ectomycorrhizas.

Ontogeny of Eucalyptus pilularis – Pisolithus tinctorius ectomycorrhizae. II. Transmission electron microscopy

Eucalyptus pilularis – Pisolithus tinctorius ectomycorrhizae were synthesized in growth pouches, and ultrastructural features of the two symbionts were documented during ontogeny. In the root cap – meristem zone, fungal hyphae envelop the root cap and penetrate between root cap cells. These fungal hyphae have numerous organelles and nuclei, some of which are in mitosis. The inner mantle hyphae in this zone and in the pre-Hartig net zone are heterogeneous in cytoplasmic content and are separated from the epidermis by an electron-dense layer. In the young Hartig net zone, hyphae penetrate between epidermal cells which contain electron-dense vacuolar deposits. In this zone and in the older Hartig net zone, hyphae do not penetrate beyond the epidermis and therefore a paraepidermal Hartig net is formed. The outer cortical layer develops as a hypodermis with suberized cell walls. The root–fungus interface consists of Hartig net hyphae which form a labyrinthine wall system and epidermal cells which are enlarged radially and contain electron-dense vacuolar deposits.

The root–fungus interface as an indicator of symbiont interaction in ectomycorrhizae

Seedlings of Alnuscrispa (Ait.) Pursh, Alnusrubra Bong., Eucalyptuspilularis Sm., and Betulaalleghaniensis Britt. were grown in plastic pouches and subsequently inoculated with Alpovadiplophloeus (Zeller & Dodge) Trappe & Smith (two different strains), Pisolithustinctorius (Pers.) Coker & Couch, and Laccariabicolor (R. Mre) Orton, respectively, to form ectomycorrhizae insitu. Alnus seedlings were inoculated with Frankia prior to inoculation with the mycosymbiont. The interface established between A. crispa and A. diplophloeus was complex, involving wall ingrowth formation in root epidermal cells and infoldings in Hartig net hyphae. Alnusrubra – A. diplophloeus ectomycorrhizae had an interface lacking epidermal cell wall ingrowths but with infoldings in Hartig net hyphae. The interface between E. pilularis –. tinctorius consisted of branching Hartig net hyphae between radially enlarged epidermal cells lacking wall ingrowths. Ectomycorrhizae between B. alleghaniensis and L. bicolor developed unique interfaces with radially enlarged epidermal cells near the apical meristem, which synthesized dense vacuolar deposits. Very fine branchings occurred in Hartig net hyphae.

SCANNING ELECTRON MICROSCOPY OF CRYOFIXED MYCORRHIZAS OF SITKA SPRUCE, PICE A SITCHENSIS (BONG.) CARR.: A COMPARISON WITH CRITICAL POINT‐DRIED MATERIAL

SummaryCritical point‐drying is the standard method of preparation for scanning electron microscope studies of mycorrhizas. However, a number of recent studies on various biological specimens have shown disadvantages associated with this technique. These can be overcome by cryofixation, where specimens are frozen in liquid nitrogen and viewed in the frozen‐hydrated state. Three distinct types of mycorrhiza of Sitka spruce [Picea sitchensis (Bong.) Carr.] were critical point‐dried or cryofixed, and were examined for differences in the preservation of both surface features and internal morphology after fracturing or cutting the material transversely.Surface features were better preserved in frozen‐hydrated mycorrhizas than in critical point‐dried ones. Surface cementing matrix seen in the former was partially or entirely removed by critical point‐drying. Some distortion of both fungal and plant cells also occurred after the latter treatment, and cell contents retained in frozen‐hydrated specimens were often lost after critical point‐drying. Fractured frozen‐hydrated material revealed surface features of the cortical cells and lobed Hartig net hyphae.

Structure and ontogeny of Alnus crispa – Alpova diplophloeus ectomycorrhizae

Alnus crispa (Ait.) Pursh seedlings were grown in plastic pouches and inoculated with Frankia to induce nodules and subsequently with Alpova diplophloeus (Zeller & Dodge) Trappe & Smith to form ectomycorrhizae. The earliest events in ectomycorrhiza formation involved contact of the root surface by hyphae, hyphal proliferation to form a thin mantle, and further hyphal growth to form a thick mantle. Structural changes in the host, the mycosymbiont, and the fungus–epidermis interface were described at various stages in the ontogeny of ectomycorrhizae. Fungal hyphae in contact with epidermal cells in the regions of intercellular penetration and paraepidermal Hartig net developed numerous rough endoplastic reticulum cisternae. In more proximal regions of the mycorrhiza, these gradually became fewer in number and smooth. A complicated labyrinthine wall branching system also developed in the fungus in these regions. Concurrently, epidermal cells formed wall ingrowths in regions adjacent to Hartig net hyphae. There was a gradient in the formation of these epidermal transfer cells as the mycorrhiza developed, and an additional deposition of secondary cell wall over the wall ingrowths occurred as transfer cells senesced. Nonmycorrhizal control roots did not develop epidermal wall ingrowths. Electron-dense material, which was also autofluorescent, was deposited in the outer tangential walls of the exodermis contiguous to the paraepidermal Hartig net.

A structural study of the interaction between the ectomycorrhizal fungus Pisolithus tinctorius and Pinus strobus roots

Stages in ectomycorrhizal development between the fungus Pisolithus tinctorius (Pers.) Coker & Couch and short roots of Pinus strobus L. were followed in growth pouches. Short roots from preinoculation through Hartig net formation were processed for light and electron microscopy. Fungal hyphae approaching the surface of roots have Thiéry-positive substances in their modified walls and in lomasomes, indicating the possibility of extracellular polysaccharide secretion. Hyphae grow between and beneath the flattened, tannin-filled superficial root cells and subsequently into the intercellular region of the cortex. A Hartig net several hyphae wide is formed, isolating the cortical cells from each other. Plasmodesmata were not observed in these cortical cells. Middle lamella material is always present around the intercellular hyphae, suggesting limited digestion by the hyphae. Cortical cell cytoplasm becomes necrotic in regions of mature Hartig net formation.

The Fine Structure of the Mycorrhizas of Pinus Radiata D. Don

The fine structure of one of two common types of mycorrhiza formed by Pinus radiata D. Don is described. The results showed inter alia that the ability of a fungus to form mycorrhiza depended on its tolerance to polyphenolic compounds in the so-called tannin layer of the cortex. Hartig net formation is a process in which the invading fungus lysed the middle lamellae and then separated the cells of the host by mechanical action. Evidence of the transfer of carbohydrate from the cortical cells of the host into the fungus in the Hartig net was obtained. Typical dolipore septae were observed both in the mantle and Hartig net hyphae. A new and hitherto undescribed structure was observed in the fungal partner.