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Section 4.

ECTOMYCORRHIZAS

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Written by Mark Brundrett
CSIRO Forestry and Forest Products
Illustrations are by the author unless otherwise stated
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Index

  1. Introduction
  2. Looking at ECM
    1. Whole Plants
    2. The Dissecting Microscope
    3. The Compound Microscope
  3. Structures and Developmental Stages
    1. Root Systems
    2. Soil Hyphae
    3. Root Contact and Hyphal Proliferation
    4. Mycorrhizal Roots
    5. The Hartig Net
    6. Fruit Body Production
  4. ECM Fungi
  5. Host Plants
    1. Dual ECM/VAM Associations
  6. Terminology

A. Introduction

Ectomycorrhizal associations (abbreviated as ECM) are mutualistic associations between higher fungi and Gymnosperm or Angiosperm plants belonging to the families listed here. As is shown below, ECM associations consist of a soil mycelium system, linking mycorrhizal roots and storage or reproductive structures. Ectomycorrhizal roots (which have also been called ectotrophic associations or sheathing mycorrhizas) are characterised by the presence of a mantle and Hartig net, but both these structures may not be well developed. Detailed descriptions of ECM root morphology have been published elsewhere (eg. Kottke & Oberwinkler 1986, Massicotte et al. 1987).

Ectomycorrhizal associations are formed predominantly on the fine root tips of the host, which are unevenly distributed throughout the soil profile, being more abundant in topsoil layers containing humus, than in underlying layers of mineral soil (Meyer 1973, Harvey et al. 1976). ECM fungi can make a significant contribution to the biomass of forest ecosystems (Marks et al. 1968, Vogt et al. 1981, Hunt & Fogel 1983). The hyphae of mycorrhizal fungi are widely distributed through the soil and are considered to make a large contribution to nutrient uptake and cycling in many ecosystems.

B. Looking at ECM

These diagrams show increasingly magnified views of typical angiosperm and pine mycorrhizal root systems with characteristically branched short roots and ectomycorrhizal structures. Information on the methods required to see mycorrhizal structures is provided in Section 8.
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Level 1. Whole Plants

Level 1 (15KB)

Mycorrhizal roots and the associated networks of hyphae are a major component of most soils, but structures produced by ECM fungi may be hard to see with the naked eye (their size is exaggerated in this diagram).

Samples of roots or soil can be collected, processed and viewed with a microscope as described below to detect mycorrhizas.
The fruit bodies of ECM fungi normally are conspicuous structures, that often can be identified by macroscopic features.
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Level 2. The Dissecting Microscope

Level 2 (8KB)
The medium level of magnification provided by a dissecting microscope allows hyphae and mycorrhizal roots to be seen. These can be observed in situ or by washing soil from roots very gently.
Observations of fresh roots with a dissecting microscope normally allow ECM roots to be identified and quantified. However, some roots must be cleared in hot alkali to make them transparent and stained with a dye (Trypan blue or Chlorazal black E) that binds to fungal hyphae to identify mycorrhizas.

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Level 3. The Compound Microscope

Level 3 (18KB)
The higher level of magnification provided by a compound microscope allows mycorrhizal structures within roots to be seen.
Thin sections of roots reveal structural details of mycorrhizas. Whole roots or root sections can be cleared and stained to remove root pigments and show fungus hyphae.
Microscopic observations can confirm the identity of fungi and reveal their spores.

C. Structures and Developmental Stages

Ectomycorrhizas form by synchronised growth by host roots and compatible fungi when environmental conditions are favourable. The sequence of events that results in ECM formation has been described in many studies (Chilvers & Gust 1982, Kottke & Oberwinkler 1986, Massicotte et al. 1987). These events are summarised below.
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1. Root Systems

Most plants with ECM have roots with a modified lateral root branching pattern. This pattern, which is called heterorhizy, consists of short mycorrhizal lateral roots (called short roots) supported by a network of long roots. The long and short roots in heterorhizic root systems are fundamentally similar in structure, but short roots normally grow much more slowly than long roots (Wilcox 1964, Kubíková 1967). The restricted growth of short roots may be necessary to allow ECM fungi time to form an association, since these fungi have difficulty colonising more rapidly growing roots (Chilvers & Gust 1982). Thus, trees with ECM would require slow growth of some of their lateral roots, and in time, this process would result in the evolution of separate, genetically distinct long and short roots. The long roots of many species rapidly develop a periderm, which would prevent them from forming mycorrhizas.

Ectomycorrhizal short roots (16KB) Example of ECM short roots (arrows) of birch (Betula alleghaniensis), an angiosperm tree. The mycorrhizal short roots are thicker than other laterals of the same order due to the mantle and Hartig net. These structures are explained below.

(grid = 1 mm)

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2. Soil Hyphae

Diagram of ECM fungus hyphae (6KB) Mycorrhizal fungi produce a hyphal network in soils. This network consists of individual strands of hyphae and/or relatively undifferentiated bundles of hyphae called mycelial strands (Agerer 1991). Some fungi can produce rhizomorphs, which contain specialised conducting hyphae, or sclerotia, which are resistant storage structures. Soil hyphae function by acquiring nutrients re-allocating resources for reproduction or mycorrhizal exchange and by functioning as propagules to allow survival and spread of the fungus.
Images of bidirectional transport in Pisolithus hyphae are provided by Ann Ashford and the Mycorrhizal Research Group of the School of Biological Science at the University of New South Wales.

Pisolithus hyphae and ECM (9KB) Hyphae of a Pisolithus species growing on a Petri dish forming mycorrhizas with Eucalyptus grandis (arrows).

(Bar = 1 cm)
Photo courtesy of Treena Burgess, Burnie Dell & Nick Malajczuk (see Burgess et al. 1994)

ECM root with black hyphae (7KB) ECM short roots (S) of Eucalyptus globulus with a Cenococcum-like mycorrhiza that has relatively thick black radiating external hyphae (arrow).


(Magnification = approx. 40x)
Photo courtesy of Nick Malajczuk

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3. Root Contact and Hyphal Proliferation

Hyphae contact, recognise and adhere to root epidermal cells near the apex of young, actively growing, high-order, lateral root. These are called short roots because they normally have limited longitudinal growth. These early stages in the establishment of ECM associations are illustrated by two scanning electron microscope (SEM) images taken during a study of the development of ECM of Pinus strobus by Piché et al. (1983ab). These show attached hyphae on the root surface 1-2 days after first contact with the root. They found a differentiated mantle and the Hartig net were present 2-4 days after root contact by the fungus.

SEM of early stage of pine ECM (15KB) Early stage of colonisation of pine short root by Pisolithus tinctorius. Hyphae (arrows) have contacted the root and are starting to proliferate on its surface near the apex (A).


(Bar = 10 um)
Photo courtesy of Yves Piché & Larry Peterson

SEM of later stage of pine ECM (21KB) SEM image showing the next stage of pine root colonisation by Pisolithus tinctorius. Mantle hyphae (arrows) have formed a dense covering on the root surface (arrows).


(Bar = 100 um)
Photo courtesy of Yves Piché & Larry Peterson

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4. Mycorrhizal Roots

After ECM associations are established mycorrhizal short roots continue to grow by elongation and branching. Pine roots with ECM usually are easily recognised due to their dichotomous branching patters. The size, colour, texture and branching patterns of ECM roots vary with different host-fungus combinations. The images below are from a host-fungus compatibility study (Malajczuk et al. 1982) and only begin to illustrate these variations. Pictures showing variations in eucalyptus ECM roots are presented in Section 7.

Pine Suillus ECM (9KB) Ectomycorrhizal association synthesised under sterile conditions between Pinus radiata and Suillus brevipes. These dichotomously branched mycorrhizal short roots increase in age from left to right.

(Magnification = approx. 12x)
Photo courtesy of Nick Malajczuk,
Randy Molina & Jim Trappe

Pine Amanita ECM (13KB) Pinus radiata and Amanita muscaria ECM synthesised under sterile conditions. This association has highly branched short roots with many root tips (arrows).

(Magnification = approx. 24x)
Photo courtesy of Nick Malajczuk,
Randy Molina & Jim Trappe

Eucalyptus Astraeus ECM (10KB) Eucalyptus maculata and Astraeus pteridis association synthesised under sterile conditions with relatively unbranched ECM (arrows) and attached mycelial strands (star).

(Magnification = approx. 10x)
Photo courtesy of Nick Malajczuk,
Randy Molina & Jim Trappe

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5. The Hartig Net

Hyphae penetrate between host cells and branch to form a labyrinthine structure called the Hartig net. Host responses may include polyphenol production in cells, phenylpropanoid accumulation and the deposition of secondary metabolites in walls (Weiss et al. 1997, Ling-Lee et al. 1977, Brundrett et al. 1990). Angiosperms with ECM such as Eucalyptus, Betula, Populus, Fagus, Shorea, etc. usually have a one cell layer Hartig net which is confined to the epidermis (Alexander & Hogberg 1986, Massicotte et al. 1987). This contrasts with the typical situation in Gymnosperms such as Pinus, where Hartig net hyphae extend deep into the cortex (Harley & Smith 1983, Kottke & Oberwinkler 1986). Examples of typical Angiosperm and Gymnosperm ECM roots are shown below. Structural characteristics of host roots, particularly of cells in the hypodermal layer are thought to restrict ECM fungus hyphae to the epidermis in most Angiosperms (Ling-Lee et al. 1977, Brundrett et al. 1990). Hyphal penetration in Gymnosperms may also be stopped by inner-cortex wall features in some cases (Brundrett et al. 1990).

The active mycorrhizal zone occurs several mm behind the root tip (as a result of the time required for mycorrhizal formation), but Hartig net hyphae senesce (as indicated by ultrastructural changes) in older regions further from the root tip (Massicotte et al. 1987). Consequently, Hartig net activity depends on root age and root growth. The mantle in older roots generally persists long after associations become inactive. Older ECM roots probably function as storage structures and propagules.

The images below are hand sections of Canadian forest trees cleared and stained with Chlorazol black E and viewed with interference contrast microscopy (Brundrett et al. 1991).

Low magnification of pine ECM (12KB) Cross section of Pinus strobus (White pine) ECM short root with thick mantle (M) and Hartig net hyphae (arrows) have enveloped several layers of cortex cells.


(Magnification = 215x)

Hartig net of pine (11KB) Higher magnification view in the Pinus strobus ECM short root shown above. This image shows how Hartig net hyphae (arrows) envelop cortex cells (C).


(Magnification = 540x)

Hartig net of Picea (13KB) Cross section of a Picea glauca ECM with labyrinthine Hartig net hyphae (arrows). Note the tannin-filled epidermal cells in the inner mantle.

(Magnification = 540x)
Abbreviations: C = cortex cell,
E = epidermal cell

Hartig net of Populus (15KB) Populus tremuloides ECM root cross section showing labyrinthine Hartig net hyphae (arrows) around elongated epidermal cells. This complex hyphal branching pattern is considered to increase the fungal surface area in contact with the root.

View larger image (37 KB)

(Magnification = 540x)
Abbreviations: C = cortex cell,
E = epidermal cell, M = mantle,
En = endodermis

Longitudinal section of Abies ECM (13KB) Longitudinal section of an Betula papyrifera ECM root showing how epidermal cells have become radially elongated to increase the area available for Hartig net hyphae (arrows).

(Magnification = 540x)
Abbreviations: C = cortex cell,
E = epidermal cell, M = mantle

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6. Fruit Body Production

The hyphal network that interconnects the structures produced by mycorrhizal fungi in soils can also produce fungal fruit bodies used for reproduction. Fruit bodies grow from primordia, which typically only form at certain times of the year, when environmental conditions are favourable. However, some fungi will fruit under mycorrhizal plants growing in pots at any time of the year if they have sufficient resources.

Fungi fruiting under a mycorrhizal eucalypt (15KB) Fruit bodies of the ECM fungus Laccaria produced under an inoculated eucalyptus seedling grown in a pot of pasteurised soil for several months.

(Magnification = approx. 1x) Photo courtesy of Neale Bougher

D. ECM Fungi

The reproductive structures of ECM fungi include epigeous fungi (mushrooms, puffballs, coral fungi, etc.) and subterranean structures (hypogeal fungi which are called truffles or truffle-like fungi). The majority of fungi that form ECM associations are Basidiomycetes, with some Ascomycetes and a few Zygomycetes (Molina et al. 1992). It has been estimated that 6000 or mores species of fungi form ECM associations with approximately 10% of the Angiosperms and many Gymnosperms (Trappe 1987). A list of known Australian ECM fungi is provided and important genera which associate with Eucalyptus species are illustrated.

E. Host Plants

Trees with ECM associations are dominant in coniferous forests, in cold boreal or alpine regions, and many of the broad-leaved forests in temperate or mediterranean regions, but they also occur in some tropical or subtropical savanna or rain forests habitats (Meyer 1973, Alexander & Hogberg 1986, Brundrett 1991). The majority of ECM hosts are trees, or shrubs (see Table), but associations are formed by a few herbaceous plants, including Kobresia (Cyperaceae), Polygonum (polygonaceae) and Cassiope (Ericaceae) species found in alpine/arctic regions (Kohn & Stasovski 1990, Massicotte et al. 1998). Some Australian herbaceous plants in the families Goodeniaceae, Asteraceae and Stylidiaceae have also been reported to form ECM, but this is controversial (Kope & Warcup 1986). Ectomycorrhizal families and genera are listed in the Table below, which contains data from Alexander & Hogberg (1986) and Harley & Harley (1987) and Brundrett et al. (1996).

Families and genera of plants with typical ectomycorrhizal associations
Family

Genera

Betulaceae Alnus, Betula, Carpinus, Ostrya, Ostryopsis
Caesalpiniaceae Anthonotha, Afzelia, Berlinia, Brachystegia, Eperua, Gilbertiodendron, Intsia, Isoberlinia, Julbernardia, Microberlinia, Monopetalanthus, Tetraberlinia
Casuarinaceae Allocasuarina (Cassuarina)
Cistaceae Helianthemum, Cistus, Tuberaria
Corylaceae Corylus
Cyperaceae Kobresia (herb)
Dipterocarpaceae Anisoptera, Dipterocarpus, Hopea, Marquesia, Monotes, Shorea, Vateria
Ericaceae Cassiope
Euphorbiaceae Marquesia, Uapaca, Ampera, Poranthera
Papilionaceae (Fabaceae) Gastrolobium, Gompholobium, Jacksonia, Mirbelia, Oxylobium, Pericopsis and other Australian peas
Fagaceae Castanea, Castanopsis, Fagus, Nothofagus, Quercus
Gnetaceae Gnetum
Meliaceae Owenia
Mimosaceae Acacia
Myrtaceae Allosyncarpia, Agonis, Angophora, Baeckea, Eucalyptus, Leptospermum, Melaleuca, Tristania and other Australian plants
Nyctaginaceae Neea, Pisonia
Pinaceae Abies, Cathaya, Cedrus, Keteleeria, Larix, Picea, Pinus, Pseudolarix, Pseudotsuga, Tsuga
Polygonaceae Polygonum
Rhamnaceae Pomaderris, Trymalium
Rosaceae Dryas
Salicaceae Populus, Salix
Tiliaceae Tilia
Notes: Gymnosperms. Families with many VAM plants. Families with many nonmycorrhizal plants. Excluded families that appear in some lists, but have not been well documented or have atypical associations: Aceraceae, Aquifoliaceae, Asteraceae, Bignoniaceae, Campanulaceae, Brassicaceae, Caprifoliaceae, Caryophyllaceae, Cornaceae, All Ferns, Goodenaceae, Lauraceae, Myricaceae, Oleaceae, Plantanaceae, Rubiaceae, Saxifragaceae, Stylidiaceae, Thymeliaceae, Ulmaceae, Vitaceae.

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1. Dual ECM/VAM Associations

Surveys of the mycorrhizal literature have established that plants within a genus usually have the same type of mycorrhizas (ECM, VAM, etc. or remain nonmycorrhizal) and these relationships are generally also consistent within a family (Harley & Harley 1987, Newman & Reddell 1987, Brundrett & Abbott 1991). This high correlation between plant phylogeny and mycorrhizal relationships has been observed for families with ECM, as well as those containing species that are usually nonmycorrhizal, but there are also many exceptions (Harley & Harley 1987, Testier et al. 1987, Brundrett 1991, Molina et al. 1992).

Australian plants with ECM associations usually also have some VAM in their roots, but little is known about the relative importance of these associations. Australian plants reported to have dual VAM/ECM associations include key species used in plantation forestry belonging to the genera Casuarina, Allocasuarina (Casuarinaceae), Eucalyptus, Melaleuca (Myrtaceae) and Acacia (Mimosaceae) (Brundrett et al. 1996). Plants with dual ECM/VAM associations are less often reported from other parts of the world (Brundrett 1991), but there are exceptions such as Alnus, Populus, Salix and Uapaca (Lodge & Wentworth 1990, Zhao Zhong, 1995; Moyersoen & Fitter, 1999).

Many reports of VAM in roots of species which normally only have ECM, concern hyphae and vesicles, but not arbuscules (Vozzo & Hacskaylo 1974, Malloch & Malloch 1981, Harley & Harley 1987, Cázares & Trappe 1993). It is unlikely that these reports result from mis-identification of the fungus, since VAM-fungus hyphae and vesicles have a characteristic appearance. However, VAM fungi also colonize a variety of substrates, including rhizome scales and senescing roots of nonmycorrhizal species (St. John et al. 1983, Brundrett & Kendrick 1988, Cázares & Trappe 1993). Thus it is probable that some reports of VAM hyphae in non-host roots represent such saprophytic activity. These roots may not be able to completely exclude VAM hyphae, given the ubiquitous presences of their inoculum in soils (Harley & Smith 1983).

F. Terminology

This glossary defines important terminology used to describe ectomycorrhizas (ECM).
Mantle
layers of fungal hyphae covering the root surface.
Hartig net
Labyrinthine network of specialised fungus hyphae, with frequent branching (or wall ingrowths) that forms a layer between the walls of adjacent root epidermal or cortex cells. This is considered to be the major site of nutrient exchange between the fungus and host plant.
Heterorhizy
Root system with distinct long and short elements, resulting from reduced longitudinal growth by fine laterals.
Short roots
These are roots with ECM with reduced apical growth and more frequent branching, resulting in heterorhizy.
Long roots
The lateral roots which bear ECM short roots. These often undergo early secondary growth.
Dichotomous branching
This is a distinctive form of branching of the ECM short roots of some Gymnosperm trees (e.g. Pinus species). These bifurcations result in two equal branches and may result in a cluster of branches with an even number of root tips.
Pinnate branching
Also called sympodial branching, is unequal branching of mycorrhizal lateral roots with perpendicular side branches.
Extraradical hyphae
Hyphae of fungi growing in the soil are also called mycelia or the fungus thallus. They may extend outwards from the fungal mantle. Hyphae strands into the soil. These initiate mycorrhizal associations, acquire soil nutrients, etc.
Mycelial strands and rhizomorphs
Interwoven hyphae that function as transport conduits and spread the association. Rhizomorphs contain specialised types of hyphae.
Fruitbodies
Thes eare also called sporocarps, basidiocarps, ascocarps, mushrooms, truffles, etc. They are relatively large reproductive structures formed by Basidiomycetes or Ascomycetes which form sexual basidiospores or ascospores respectively. These develop from primordia produced by the mycelial system.
Sclerotia
Storage structures produced in soil by some fungi, comprised of compact fungal tissue, which is often highly melanised.
Other spores
Small asexual spores (conidia) which function as propagules, may be produced by some mycorrhizal fungi.

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This page was last revised August 3 1999


Start
Here

Intro-
duction

Roots

VAM

Top of
this page

Roles

Australian
Plants

Eucalypt
Associates

Methods

Refer-
ences