Mycorrhizal Symbiosis

By Sally E. Smith and David J. Read

In nature, the roots of most plants are colonized by symbiotic fungi to form mycorrhiza, which play a critical role in the capture of nutrients from the soil, and therefore in plant nutrition. Thirteen years have passed since the publication of the First Edition of Mycorrhizal Symbiosis, the book that has been generally acclaimed as the most definitive work on this fascinating topic. The Second Edition co-authored by Professor Sally Smith and Professor David Read has been completely rewritten to cover the significant advances in our understanding of this field.

Key Features* Separate accounts of major mycorrhizal types, highlighting structure, development, physiology and ecology* Integrative treatment, covering nutrient transport, roles of mycorrhizas in ecology, applications in man-made environments, and interactions with pollutants* In depth treatment of evolutionary and developmental aspects, plus closer examination of external mycelium, and transport processes* Appreciation of diversity of form and function within major mycorrhizal types, and its importance in ecosystems

• Language: English
• Publisher: Academic Press
• Release date: Oct 25, 1996
• ISBN: 9780080537191

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plants.

Section 1

Vesicular-arbuscular Mycorrhizas

1

The symbionts forming VA mycorrhizas

Introduction

Vesicular-arbuscular (VA) mycorrhizas are the most common underground sym-biosis. They are formed. in the roots of an enormously wide variety of host plants by aseptate, obligately symbiotic fungi in the order Glomales (Zygomycotina). The plants include angiosperms, gymnosperms and pteridophytes, which all have true roots, as well as the gametophytes of some mosses, lycopods and Psilotales, which do not (see Pocock and Duckett, 1984, 1985; Peterson et al., 1981). It seems highly likely that the fungi had their origins between 353 and 462 million years ago and that the symbiosis is similarly ancient and was probably important in the colonization of land by vascular plants (Simon et al., 1993).

The name ‘vesicular-arbuscular’ is derived from characteristic structures, the arbuscules (see Fig. 1.1) which occur within the cortical cells, and vesicles which occur within or between them. A VA mycorrhiza has three important components: the root itself, the fungal structures within the cells of the root and an extraradical mycelium in the soil. The last may be quite extensive under some conditions but does not form any vegetative pseudoparenchymatous structures comparable to the fungal sheath typical of ectomycorrhizas. A few of the fungi, however, do form sporocarps with limited amounts of sterile mycelium (Fig. 1.2; Plate 1). The majority (about 80%) of species presently described form both arbuscules and vesicles. The remainder do not form vesicles and should therefore strictly be called ‘arbuscular’ mycorrhizal fungi. In some plant species the fungus grows intercellularly and intracellular hyphal coils are restricted to a few cell types only; in others, the same fungi may form abundant coils in the cortical cells of the root and in these mycorrhizas the development of arbuscules may be reduced. Because the characteristic fungal structures develop within the root and changes in the rates of root growth and branching are discernible only by detailed comparison with non-colonized plants, it is usually impossible to tell whether or not a root system is mycorrhizal without staining and microscopic examination. A few plants, such as Allium and Zea, do synthesize a yellow pigment when colonized but this is not sufficiently frequent to be a generally useful diagnostic character.

Figure 1.1 A mature arbuscule of Glomus mosseae within a cortical cell of Allium porrum (leek). The arbuscule has grown from a well developed intercellular hypha (arrow). From Brundrett et al. (1984), with permission.

Figure 1.2 (a) Spore and subtending hypha of Glomus invermoius. Spore diameter approximately 75 μm. (b) Sporocarp of Sclerocystis rubiformis (slightly squashed). Diameter of individual spores approximately 45 μm. From Hall and Abbott (1981), with permission.

Plate 1 (a) Spores and subtending hyphae of Gigaspora margarita, approximate diameter 400–450 μm. (b) Spore of Acaulospara laevis (arrowed), attached to the neck of the sporiferous saccule (s). Spore diameter approximately 190–210 μm. (c) Spores of Scutel-lospora nigra, approximate diameter 300μm. Photographs courtesy of V. Gianinazzi-Pearson.

VA mycorrhizas were first recognized and described in the last decades of the nineteenth century. Their widespread occurrence and common presence in plants of many phyla in most parts of the world, especially in the tropics, was realized very soon, but very little functional information was learned about them until the mid-1950s. The early work was reviewed by Rayner (1927) but she discussed the nature of the fungal symbiont without reaching a convincing conclusion. Indeed, much of the effort put into research on this type of mycorrhiza during the 1920s and 1930s was vitiated by the relative ease with which fungal inhabitants of the root surface and of senescing cells could be isolated into culture and the difficulty, still unsurmounted, of isolating the fungal symbionts themselves. Almost all writings about the identity of the fungus until 1953 may be ignored, except for those of Peyronel who, in 1923, showed that the hyphae of the endophyte might be traced to the sporocarps of species of Endogonaceae in the surrounding soil, and of Butler (1939) who, in an influential review, agreed that the fungi called Rhizophagus were almost certainly imperfect members of the Endogonaceae, which then included all fungi now transferred to the orders Glomales and Endogonales. The work of Mosse (1953), which showed convincingly that mycorrhizal strawberry plants were colonized by a species of Endogone (later transferred to Glomus), may be said to have heralded the modern period. Soon Mosse, Baylis, Gerdemann, Nicolson, and Daft and Nicolson greatly extended these early observations and demonstrated by inoculation that endogonaceous (glomalean) fungi were symbiotic with many kinds of plants forming the so-called phycomycetous or VA endomycorrhizas.

A major milestone was reached in 1974 with a sucessful symposium on endomycorrhizas at which a number of key ideas were developed for the first time. Many of the papers presented at that meeting remain classics (see Sanders et al., 1975). At about this time the first formal Linnean classification of the species was developed (Gerdemann and Trappe, 1974) and many general aspects of development and function of this widespread symbiosis were outlined and discussed in the first edition (Harley and Smith, 1983). While more recent work has extended and confirmed the generalizations made at that time, there has also been an increased appreciation of the diversity to be found in VA mycorrhizas and an important new emphasis on the cellular and molecular interactions between the symbionts (see Giovannetti and Gianinazzi-Pearson, 1994; Smith, 1995).

In brief, VA mycorrhizal fungi have been recognized as ecologically obligate symbionts of a very wide range of plant species. The symbiosis is biotrophic and normally mutualistic, the long-term compatible interaction being based on bidirectional nutrient transfer between the symbionts. Unlike the biotrophic parasitic symbioses, these associations show a very low degree of taxonomic specificity, a point which will be developed later in this chapter. Because the fungi have very limited capacity for growth from propagules such as spores or from the vesicles or hyphae within root fragments, special methods have had to be adopted to maintain pure strains for experimental or taxonomic purposes. As far as is possible, isolates from single spore types are grown in ‘pot culture’ on the roots of plants, so that their spore characteristics, mode of colonization and effects on plant growth can be followed by sequential sampling. However, in many cases spore collections from soil form the only basis for taxonomic study.

Fungi

Systematics

Until relatively recently the causal organisms of VA mycorrhizas were classified in the family Endogonaceae of the order Endogonales. The regular association of the very large spores and sporocarps of members of this family with VA mycorrhizal roots was established long ago by Peyronel (1923). Only later, after the work of Butler (1939) and most especially Mosse (1953, 1956), were they recognized as the chief causal organisms of VA mycorrhizas. This recognition prompted renewed interest in the taxonomy of the family, based mainly on the development, morphology and wall structure of the globose zygospores, azygospores and chlamydo-spores, and of sporangia (see Fig. 1.2 and Plate 1).

Except for zygospores produced by the Endogonaceae (sensu stricto) it is frequently difficult for the type of spore to be determined with confidence and there is disagreement among the experts on the appropriate nomenclature. To avoid such problems we refer simply to ‘spores’, while recognizing that their development in different taxa varies. Interest in mycorrhizas has led to the realization that members of this family are among the most common soil fungi and that spores or sporocarps can be collected from almost any soil. As increasing numbers of fungi with very large spores (up to 500 μrn in diameter) were collected and described, the genus Endogone grew into an unwieldy and variable assemblage of species about which few generalizations could be made. The Linnean classification of the Endogonaceae (Gerdemann and Trappe, 1974, 1975) made no attempt to relate taxonomy to the phylogeny of the group. The family contained seven genera. Endogone sensu lato was split and only those fungi forming true zygospores were retained within Endogone sensu stricto. Some of these can be cultured and they appear either to be non-mycorrhizal saprophytes or to form ectomycorrhizas; none form VA mycorrhizas. Members of the genera Gigaspora, Acaulospora, Glomus and Sclerocystis form VA mycorrhizas; Glaziella and Modicella were considered at that time to have unknown affinities. Gerdemann and Trappe (1975) regarded their revision of the Endogonaceae as a ‘temporary solution to a difficult taxonomic problem’, but it was important because it finally put the study of VA mycorrhiza on a firm taxonomic basis. Both dichotomous (Mosse and Bowen, 1968; Hall and Fish, 1979; Hall, 1984) and synoptic (e.g. Trappe, 1982) keys have been produced to help identification. In a revision of the classification, Modicella and Complexipes (a later addition) were removed by Trappe and Schenck (1982), while Glaziella was found to have ascomycetous affinities (see Walker, 1987).

By 1993 about 150 species had been described, although early descriptions are in many cases unsatisfactory and revisions are to be expected. The species are widely distributed globally, in accordance with their probable ancient origins. The classification of VA mycorrhizal fungi is at present based almost exclusively on the structure and development of the walls of the spores, so that murographs (Fig. 1.3) are an important component of taxonomic descriptions (Walker, 1983, 1992; Morton and Benny, 1990; Morton and Bentivenga, 1994), irrespective of whether identification is the main priority or phylogenetic conclusions are sought (Morton, 1990a). There is no doubt that analyses of DNA sequences and other biochemical characteristics such as fatty acid methyl ester (FAME) profiles will complement the morphological information to a greater and greater extent (Graham et al., 1995; Morton et al., 1995).

Figure 1.3 Discrete stages in differentiation of subcellular structures, characters and character states in Gigaspora and selected Scutellospora species. (a) Murographic representation of five stages in differentiation of spores of S. heterogama. Spore wall, sw; first inner wall, iwl; second inner wall, iw2; germination shield, gs; ornamentation character state, 0. Patterns indicate characters of each structure: no pattern indicates the outer layer; vertical dashed lines, laminae; angled lines, flexible layers. (b) Murographs illustrating phenotypes of adult spores. Left, Gigaspora species and right, five species of Scutellospora. Within the genera each species is separated by different character states of the spore walls (arrows). From Morton and Bentivenga (1994), with permission.

The most recent revision of the classification represents another important stage in the development of taxonomy of VA mycorrhizal fungi by taking a phylogenetic perspective (Morton and Benny, 1990). This revised classification separates a new order, Glomales, from the Endogonales. The latter contains a single family of truly zygosporic fungi in the genera Endogone and Sclerogone. The Glomales, as we said before, is now defined as containing only those fungi for which ‘C is acquired obligately from their host plants via intraradical dichotomously branching arbuscules’ (Morton and Benny 1990). This seems a very restrictive definition as it makes a number of assumptions which have not yet been substantiated physiologically (see Gianinazzi-Pearson et al., 1991a; Smith and Smith, 1996a) and also creates some practical difficulties. It is assumed that the arbuscule is a key unifying structure and that it is the site of C acquisition by the fungi. This is by no means certain, for there is no a priori reason why intercellular hyphae or intracellular coils should not be sites of C transfer (see Chapter 14). Further, fungi with typical spore development and morphology may exist but have ‘atypical’ physiology with respect to C transfer. Variation in intraradical development may be induced by host species, or may vary with the age of the plant. Indeed, one of the greatest practical difficulties arising from the use of the arbuscule as the key feature is that in this case all descriptions of fungal species must be accompanied by evidence that the fungi will form arbuscules. This problem has already led Morton (1990a) to include only fungi for which there is visual evidence of their ability to form mycorrhizas (perhaps 40% of described species) in his review of evolutionary relationships. Notwithstanding these concerns, which have also been voiced by others (Hall, 1984; Berch, 1987; Walker, 1992), the revision is an important step forward, as it provides a classification based on particular assumptions about the phylogeny of the group which can be tested through other approaches. Steussy (1992) has emphasized the importance of independent methods, such as nucleic acid sequencing, for determining phylogenetic relationships, bearing in mind particularly the difficulties of determining which characters are primitive and which are advanced in the absence of an extensive fossil record. One point on which all agree is the need for mycorrhizal workers to keep voucher specimens of the fungi used for all investigations, whether they be taxonomic or physiological.

TheGlomales as currently defined contains two suborders, the Glomineae and the Gigasporineae (Table 1.1). In all members the vegetative mycelium and intraradical structures are aseptate and multinucleate. The spores themselves contain 1000 nuclei, or even more in some species, but the extent of heterokaryosis is unknown at present. The spores germinate and produce limited mycelium in the absence of host plants. At one stage it was thought that nuclear division might not occur until a host plant had been successfully colonized and that this might go some way to explaining the failure of the fungi to grow for prolonged periods in axenic culture (Burggraaf and Beringer, 1987, 1989). Two independent lines of investigation have now made it clear that DNA replication and nuclear division do occur in germ tubes of Gigaspora margarita (Bianciotto and Bonfante, 1992; Becard and Pfeffer, 1993). However, no evidence for the occurrence of nuclear fusion and meiosis has been obtained, so it would appear that the parasexual cycle does not operate at this stage of the life cycle of this fungus, if at all. Zygospore formation has been briefly described for one species only, Gigaspora decipiens, and exchange of nuclei via anastomoses between species and, indeed, between different isolates of the same species, is likely to occur at a low rate despite the fact that the mycelium of a single isolate anastomoses frequently (Tommerup, 1988; Tommerup and Sivasithamparam, 1990).

Table 1.1

Ordinal and family structure of the Endogonales

The features of the orders are from Morton and Benny (1990) although the inclusion does not indicate agreement (see text). Numbers in parentheses after generic nouns are the numbers of species in each genus taken from Walker and Trappe (1993). Spelling of the names is also from Walker and Trappe (1993).

It therefore seems likely that the members of the Glomales may be asexual organisms. We must assume that mutation and possibly heterokaryosis provide the main bases for the variation necessary to permit adaptation to environmental change and continuing evolution. Certainly, variations in DNA sequences have been revealed by amplification of genomic DNA using short arbitrary primers (random amplified polymorphic DNA-polymerase chain reaction; RAPD-PCR). These RAPDs show similarities of between 77% and 89% for different spores from the same (Rothamsted) line of Glomus mosseae, 60–76% similarity for spores of the same species from the same geographical region, 44–64% for spores of the same species from different regions and only 9–29% similarity between different species (Wyss and Bonfante, 1993). Considerable variation has also been revealed by isozyme banding patterns, which did not necessarily coincide with species defined by spore morphology (Hepper et al., 1988; Rosendahl, 1989). The relative contributions to this variation made by mutation and exchange of genetic material are not known at present and more work on the potential for exchange, for example within the roots of plants colonized by mixed infections, might be productive. With the rapid adoption of methods to analyse DNA sequences in VA mycorrhizal fungi we are likely to see considerable advances in knowledge of intra- and inter-specific variation and extent of clonality in the near future. It has been argued by Law and Lewis (1983) that in mutualistic symbioses the endobiont (in this case the fungus) will evolve away from a sexual habit because the selection pressures will be to maintain similarity to – rather than difference from – the parents. This certainly seems to fit with the situation in glomalean fungi: the important ecological niche for C acquisition is the apoplast of the root cortex, in which homeostasis exercised by the plant will be important in maintaining extremely constant environmental conditions compared with the variation likely in the soil.

The possibly asexual nature of VA mycorrhizal fungi means that the described taxa cannot be regarded as either biological or ecological species, but rather as phenetic or form species (Morton, 1990b; Walker, 1992). Apart from the use of arbuscules in the current definition of the Glomales, the vegetative structures, together with aspects of spore morphology that vary quantitatively (shape, size, colour), have been excluded from taxonomic or phylogenetic considerations. Characteristics of the vegetative stages, such as form of the entry points and branching of hyphae within the root, are variable between fungal species and can be used for recognition purposes by experienced observers (see Abbott 1982; Lopez-Aguillon and Mosse, 1987). Most of the species of fungi listed above produce ‘coarse’ colonizations in which intercellular hyphae are 5–10 μm or more in diameter. These can be easily distinguished from ‘fine’ colonizations, with very narrow hyphae (1–3 μm diameter), which have been assigned to Glomus tenuis (Hall, 1977). The small spores of the latter (diameter 10 μm) were long overlooked and its taxonomic position is still somewhat doubtful. The ‘fine endophyte’ is, however, extremely common in many soils and the problems that it poses are not only taxonomic but also ecological and physiological.

Fossil History and Phylogeny of Glomalean Fungi

Fossils resembling the spores of VA mycorrhizal fungi have long been recognized and date from as early as the Silurian (440–410 million years BP). While these records are doubtful or possibly the result of contamination of samples, the extensive records of both spores and structures from within plant axes or decaying plant material from the famous Rhynie chert flora (Kidston and Lang, 1921) are much more satisfactory and provide compelling evidence for the existence of symbiosis between plants and Glomus-like fungi as early as 410–360 million years BP. Recent re-examination of the Rhynie chert has revealed arbuscules within the protosteles of Aglaophyton major, which should leave us in no doubt that VA or glomalean mycorrhizas had evolved by that time (Remy et al., 1994; see Fig. 1.4a). The significance of these fossils was noted by Nicolson (1975) and subsequentlyPirozynski and Dalpe (1989) have provided a critical review of the geological history of the group. This shows continuous occurrence of Glomus- like structures into the quaternary period and the occurrence of Glomus- and Sclerocystis-like spores (as well as intraradical colonization, including arbuscules) from silicified peat from the Triassic deposits in the Antarctic (Stubblefield et al., 1987a,b,c; Fig. 1.4b,c,d). Unfortunately, no reliable fossils of other glomalean taxa have been found which would shed light on phylogenetic origins of the group. Interestingly, the arbuscules in A. major (which seems to have affinities to both bryophytes and vascular plants) are delicate structures with secondary branches of 1–2 urn. They are therefore similar to present-day arbuscules, but different from the robust arbuscules found in Antarcticycas from the Triassic (compare Figs 1.1 and 1.4a,c).

Figure 1.4 Fossil VA mycorrhizas. (a) Arbuscule (A) in a cell of Aglaeophyton from the Devonian flora of the Rhynie chert. (b) Transverse section of a mycorrhizal root of Antarcticycas, from the Triassic deposits of Antarctica. Note the colonized central cortex of the root. (c),(d) Details of colonization in Antarcticycas. (c) Dichotomously branched arbuscule (A), with relatively robust branches; (d) vesicle (V). (a) From Remy et al. (1994). Copyright, National Academy of Sciences, USA., (b),(c),(d) from Stubblefield et al. (1987b), with permission.

The first approach to a classification representing phylogentic relationships has been made by Morton (1990a), using cladistic tools and assuming evolutionary significance for 27 characters used in the analysis. The basic assumptions, which as noted earlier may be questionable, are that all glomalean fungi form mutualistic associations and produce arbuscules and that these characters unite them in a monophyletic group. The remaining characters (with the exception of unexplained variations in wall staining with Trypan Blue) are based on spore characteristics that vary qualitatively and are stable and discrete. Continuously variable characters, such as spore colour and size, are not used. Application of the cladistic approach to determination of evolutionary relationships has yielded the phylogentic tree shown in Figure 1.5a, which can serve as a hypothesis for future investigations.

Figure 1.5 Phylogenetic trees for VA mycorrhizal fungi, derived from different types of information. (a) A cladogram showing taxonomic and phylogenetic divergence among genera of VA mycorrhizal fungi, based on comparative developmental sequences of the spores. Previously unpublished, courtesy of J.B. Morton. (b) Phylogenetic trees of VA mycorrhizal fungi, based on sequences of small subunit rRNA. (i), (ii), (iii) Different trees obtained by using different methods of analysis. From Simon et al. (1993). Reprinted with permission from Nature, 363, 67–69. Copyright Macmillan Magazines Ltd.

The key features are the separation of the GIomus/ScIerocystis group from Gigaspora/Scutellospora and the existence of Acaulospora/Entrophospora as a line apparently diverging from Glomus. If the assumptions on primitive and advanced characters are correct, Glomus/Sclerocystis represent the ancestral type, as shown in Figure 1.5a, with the other two major lines being more recent in origin. However, the cladistic analysis puts Scutellospora as more highly evolved than Gigaspora, a point which is disputed by Walker (1992) largely on the grounds that Scutellospora has a more restricted present-day range. There is no fossil evidence to help distinguish between these possibilities, but DNA sequence analysis indicates that Gigaspora and Glomus are in a monophyletic group (Bruns, 1992) and also puts Scutellospora ancestral to Gigaspora (Simon et al., 1993). The molecular information is very important because it provides an independent means of investigating the phylogenetic hypotheses based on cladistics. Sequences of some sections of ribosomal genes have been obtained from 12 species of glomalean fungi and from Endogone pisiformis (Endogonaceae) as an outlier. Three methods of analysing similarities in DNA sequences provided essentially similar phylogenetic trees (Fig. 1.5b) which confirm the glomalean fungi as true fungi of monophyletic origin, divided into the same three families shown by the cladisitic approach. Lipid analysis also supports the existence of three families (Sancholle and Dalpe, 1993), but analysis of the carbohydrates in the fungal walls suggests a less clear-cut phylogeny. A large number of investigations have shown that all the walls contain chitin, but the occurrence of β(1,3)glucans in members of the Glomineae and not in the Gigasporineae, suggests that only the latter group (together with Endogone) can be regarded as true Zygomycetes, while Glomus and Acaulospora are more similar to the Entomophthorales in having both chitin and β(1,3)glucan in their walls (Gianinazzi-Pearson et al., 1994b; Lemoine et al., 1995). Application of a range of techniques to evaluate the diversity to be found in the Glomales should soon sort out these apparent discrepancies (see van Tuinen et al., 1994).

The approximate dates for the origin of the group and the divergence of the major branches given by the DNA sequence data provide a link with the palaeontological information. The analysis puts the origin of the glomalean fungi via divergence from the group represented by E. pisiformis in the Devonian between 462 and 353 million years BP and the divergence between Glomaceae and the other groups in the late Palaeozoic 250 million years BP.

An origin of Glomus-like fungi at the same time as the origin of the land plants (dated at 415 million years BP) is in agreement with the fossil record and provides some support for the theories that colonization of the land by plants such as Aglaophyton (Rhynia), with restricted absorbing axes, may have been dependent on their association with mycorrhizal fungi which increased their capacity for nutrient absorption from poor soils (Pirozynski and Malloch, 1975; Raven et al., 1978; see Fig. 1.6).

Figure 1.6 Estimated dates of origin and divergence of VA mycorrhizal (VAM) fungi. From Simon et al. (1993). Reprinted with permission from Nature, 363, 67–69. Copyright Macmillan Magazines Ltd.

Host Plants

Systematics

The range of potential host plants for VA mycorrhizal fungi is extremely wide and has been responsible for the oft-quoted statement (Gerdemann, 1968) that ‘it is so ubiquitous that it is easier to list the plant families in which it is not known to occur than to compile a list of families in which it has been found’. This continues to hold good. Some members of most families of angiosperms and gymnosperms, together with ferns, lycopods and bryophytes, develop VA infections. Trappe (1987) has produced a most valuable compilation of the incidence of all types of mycorrhizas within the angiosperms, taken from published material. Records of VA mycorrhizas are to be found in all the orders from which plants have been examined and are about equally frequent in Dicotyledonae and Monocotyledonae. He stresses that only about 3% of species have actually been examined and our knowledge of the mycorrhizal status of some taxa is very poor indeed. Consequently, it can be said that about 95% of the present-day species of plants belong to families that are characteristically mycorrhizal. But it cannot be said that 95% of the world’s species are mycorrhizal: such sweeping and inaccurate statements should be avoided (see Trappe, 1987). Futhermore, only single specimens of some species have been examined, and in these cases generalizations are risky because of variations in the extent of mycorrhizal colonization between sites and at different seasons. Nevertheless, the more we look the greater the number of species that prove to be mycorrhizal. Harley and Harley (1987) have surveyed the literature on the incidence of mycorrhizas at the species level in the very-well-studied British flora. For many families, over 40% and sometimes as high as 80% of the species have been investigated, often more than once. All families listed contained mycorrhizal species, and these frequently constituted a very high proportion of the total.

Even in families widely thought to be ‘non-mycorrhizal’, such as the Polygonaceae, Juncaceae, Cruciferae and Caryophyllaceae, mycorrhizas were found, although their presence was not consistent and the colonization often sparse. As in other surveys (e.g. Newman and Reddell, 1987), some species have been recorded as occurring in both mycorrhizal and non-mycorrhizal states and members of some plant families characteristically form mycorrhizas of other types.

VA mycorrhizas are found in most herbaceous plants that have been studied (see above for exceptions) but are by no means restricted to herbs. As long ago as 1897 Janse examined 46 species of tree in Java and found them all to have VA mycor-rhiza. More recent work, reviewed by Smits (1992) and Janos (1987) confirms this. The Dipterocarpaceae appears to be the only family of tropical trees which are typically ectomycorrhizal. Otherwise, VA mycorrhizas predominate in these taxonomically diverse systems as well as in some temperate forest systems. Thus, Baylis (1961, 1962) states that VA mycorrhizas are ecologically the most important type of mycorrhiza in New Zealand forests. Whereas the Pinaceae are ectomycorrhizal, all other conifer families are dominantly VA mycorrhizal, as are most other gymnosperms, all of which are woody. Although VA mycorrhizas have often been ignored by foresters in the past, they are characteristic of such valuable trees as Araucaria, Podocarpus and Agathis as well as all the Cupressaceae, Taxodiaceae, Taxaceae, Cephalotaxaceae and the majority of tropical hardwoods. The importance of considering the appropriate mycorrhizal associates for trees used in reafforestation programmes in temperate and tropical ecosystems is now widely recognized. While most of the experimental work on VA mycorrhiza has been done with herbs, because these are easier to manage under laboratory conditions, some trees have also been used and these include Malus (apple), Citrus, Salix, Populus, Persea (avocado), Coffea, Araucaria, Khaya, Anacardium (cashew) and Liquidambar. It is certainly important to realize that mycorrhizas may be significant in nutrient absorption and in nutrient cycling of arborescent species in forest ecosystems. Work with trees and other perennials is therefore very important both from an ecological point of view and from a need to consider forest and crop production. Indeed, although work with herbs allows greater control of conditions in growth rooms, etc., the propagation of some woody species from cuttings may have great advantages in providing genetically uniform experimental material which may partly offset the long growth periods necessary for the study of long-lived plants. The extensive work on Citrus mycorrhiza by groups in California and Florida is an example of an arborescent species of economic importance being used in experiments designed both to increase crop productivity and to promote an understanding of the development and physiology of the symbiosis.

The Fossil History of Mycorrhizal Colonization

As in the case of the spores mentioned above, the long fossil history of fungal infections in the absorbing organs of plants is well recognized. The earliest known land plants did not possess true roots, but the protostelic rhizomes of Aglaophyton (Rhynia) and Asteroxylon were clearly infected by fungi (called Palaeomyces) which formed arbuscules, intercellular hyphae and vesicles like modern members of the Glomales (Kidston and Lang, 1921; Remy et al., 1994; and see Pirozynski and Dalpé, 1989; see Fig. 1.4a).

Many gymnosperm fossils with VA mycorrhizas have been identified in later Carboniferous deposits. The best known and preserved is Amyelon radicans, which again resembled the VA mycorrhizas of living gymnosperms (see Nicolson, 1975). The Triassic flora from Antarctica (250–210 million years BP) has also yielded important evidence for the development of intraradical vegetative structures, including both intercellular hyphae and arbuscules (Stubblefield et al., 1987a,b; see Fig. 1.4b,c,d). Beautifully preserved roots of Antarcticycas, containing both septate and aseptate hyphae and structures strongly resembling mycorrhizal arbuscules, vesicles and spores, have been described by Stubblefield et al., (1987a). Sections and peels of these fossils are virtually indistinguishable from presentday mycorrhizal cycad roots. It is impossible, of course, to be sure about the physiology of these fossil mycorrhizas, but if they functioned in a manner similar to present-day forms their role in colonization of the land and in subsequent plant evolution may have been considerable. The view has been put forward that the soil available to early land plants is likely to have been deficient in available mineral nutrients, so that the intervention of the fungi in their absorption might well have been important to the success of the plants invading the terrestrial environment (Baylis, 1972b; Nicolson 1975; Pirozynski and Malloch, 1975; Raven et al., 1978). In contrast, ericoid and ectomycorrhizas, which are more typical of communities growing on organic soils are envisaged as originating more recently than VA rnycorrhizas, as soil organic matter increased.

Recent worldwide surveys have greatly increased the known number of plants that form more than one kind of mycorrhiza. Both VA and ectomycorrhizas are most commonly reported in the Rosaceae and Salicaceae; they also occur in at least 10 other angiosperm families, including the Papilionaceae and Rhamnaceae, and occasionally in the Gymnospermae and Pteridophyta (Newman and Reddell, 1987; Trappe, 1987; Brundrett and Abbott 1991; see Table 1.2). In some cases VA mycorrhizal colonization has been reported on young individuals of species usually forming ectornycorrhizas, for example Eucalyptus, Pseudotsuga and Tsuga (Lapeyrie and Chilvers, 1985; Chilvers et al., 1987; Cazares and Smith, 1992, 1996). Indeed, it is possible that VA mycorrhizal fungi may have the ability to invade the underground organs of almost all land plants. Such an attribute would explain why the long coevolution of the symbionts has not resulted in the specialization of the fungi to their host range nor in taxonomic specificity of the symbioses (Table 1.3).

Table 1.2

Numbers and percentages of species of subclasses and classes of Angiospermae examined for mycorrhiza formation and percentage of examined species by type of mycorrhiza

Other: mycorrhizas formed by ascomycetes and basidiomycetes

Based on Trappe (1987).

The form of the root system is important in influencing the extent to which plants respond, in nutrient absorption and growth, to mycorrhizal colonization. Plants bearing magnolioid type roots, characterized by wide axes (up to 1.5 mm in diameter), slow growth and poor root-hair development, are frequently highly responsive, while plants with fine, rapidly growing root systems and long root hairs are not (Baylis, 1975; St John, 1980). The importance of mycorrhizas for growth and nutrient absorption will be discussed in Chapters 4 and 5. Here the important point is that the woody Magnoliales (with magnolioid root systems) which are considered to be the most primitive living angiosperms (Cronquist, 1981), being ancestral to the other dicotyledons, have a huge incidence of species forming VA mycorrhizas and relatively few which are characteristically non-mycorrhizal or bear other mycorrhizal types (see Trappe, 1987). This frequency is greater than that observed in other more advanced dicotyledonous subclasses, as can be seen in Figure 1.7 and Table 1.2.

Figure 1.7 Phyologenetic dendrogram for the subclasses of Dicotyledonae, showing the percentage of species with zygomycetous (VA) mycorrhizas (numbers in circles), asco- and basidiomycetous (ericoid and ecto-) mycorrhizas (numbers in triangles), or no mycorrhizas (numbers in squares). Many species have mycorrhizas in more than one category, so that percentages total more than 100. Reprinted with permission from Trappe (1987) in Ecophysiology of V.A. Mycorrhizal Plants. CRC Press, Boca Raton FL.

Using only those taxa for which the mycorrhizal status is known in at least 10% of the species, and using the phylogenetic classifications of Cronquist (1981), Trappe has prepared dendrograms which allow some preliminary evolutionary conclusions to be drawn. A high incidence of VA mycorrhizas appears to have been retained in the line through the Rosidae to the Asteridae, which show relatively low incidence of other, supposedly advanced, mycorrhizal types. The line to the Caryophyllidae shows a general reduction in the incidence of any type of mycorrhiza while the line through the Hamamelidae to the present day Juglandales and Fagales shows a marked increase in ectomycorrhizas. A more detailed approach is taken within these phylogenetic lines, where data are available, and they provide fascinating hypotheses which can be investigated in future collections. The present tentative conclusions agree with the fossil record in placing VA mycorrhizas as primitive and the other mycorrhizal types as more advanced. Within the Monocotyledonae all lines (considered to be parallel by Cronquist, 1981) are heavily mycorrhizal and VA mycorrhizas predominate except in the Orchidaceae (Liliidae) which have characteristic and probably advanced orchidaceo us mycorrhizas formed by Basidiomycetes (see Chapter 13).

One of the important conclusions of this work is that the non-mycorrhizal condition has evolved several times in different phylogenetic lines and may therefore have different cellular and physiological bases. Although families containing large numbers of species in which mycorrhizal colonization is characteristically absent are relatively rare, they are worth studying in their own right for mechanisms by which the fungi are excluded, as well as the means by which they acquire nutrients from soil (Lamont, 1981, 1982; Pate, 1994; Marschner, 1995; see Chapter 3).

Specificity and Extent of Colonization

So far we have confined the discussion to the potential of different taxa of plants to form mycorrhizas in field or experimental conditions, without concerning ourselves greatly with questions about whether or not a species is always mycorrhizal, how extensively the roots are colonized, or how far it may be dependent on the mycorrhizal state for growth or reproductive success. These are complex issues which are important in discussions of cellular interactions and plant–fungus specificity and compatibility, as well as of ecology.

Specificity needs to be considered at both taxonomic and ecological levels. Taxonomic specificity or host range indicates whether or not a given species of fungus can form a mycorrhizal relationship with more than one species of host or whether or not a given species of host associates mycorrhizally with more than one species of fungus. This can be extended to lower taxonomic levels, where subspecifie genetic strains of fungus may form mycorrhizas attuned in some way to the species or subspeeific genetic strains of the host. At a still finer level we need to determine whether or not there is in mycorrhizal symbioses anything resembling the genes for ‘resistance’ or ‘avirulence’ that have been recognized in some kinds of antagonistic symbioses and which have so profoundly influenced the thinking of plant pathologists.

There is no clear evidence that any absolute specificity exists between taxa of VA mycorrhizal fungi and taxa of potential host plants and it is of interest that even before modern methods were available, investigators such as Magrou (1936), Stahl (1949) and Gerdemann (1955) had reached this conclusion. In general (accepting that a few plant families do not form mycorrhizas or usually form another type of mycorrhiza), we might expect, with reasonable confidence, that a VA mycorrhizal fungus isolated from one species of host plant will colonize any other species that has been shown to be capable of forming VA mycorrhizas, thus combining wide host range with permanence of association. It is important to grasp this point clearly, because some authors (e.g. Heslop-Harrison, 1978) have stated specifically that the angiosperm root systems in association with mycorrhizal fungi exhibit a ‘high or very high’ degree of specificity. This is incorrect and based on extrapolation from work with parasitic fungal biotrophs (e.g. rusts, smuts) where a very high degree of race–cultivar specificity has evolved. Others have recognized clearly that mutualistic and parasitic associations are subject to quite different selection pressures (Brian, 1976; Vanderplank, 1978; Smith and Douglas, 1987) and we can do no better than to quote Vanderplank in this context:

‘Opposite selection pressures are clearly involved. In parasitic symbiosis the host plant benefits by mutation to resistance because this ends, for the host, an unwanted symbiosis. In mutualistic symbiosis the host loses by mutation to resistance because this ends the symbiosis. Mutations to resistance in mycorrhizal plants are eliminated by selection because they are disadvantageous; and the elimination also eliminates a major source of specificity.’

There are, however, considerable gradations in the extent to which species of plants or even genotypes within a species become colonized by mycorrhizal fungi. The converse is also true: different species or isolates of fungi colonize the roots of the same species of plant to different extents and in a few cases the range of potential partners appears so restricted as to constitute specificity. A few examples will illustrate these points. Giovannetti and Hepper (1985) tested the ability of three legume species, Medicago sativa, Hedysarum coronarium and Onobrychis viciaefolia to be colonized by four Glomus species. Using two soils of different P availability they showed that whereas M. sativa was extensively (though variably) colonized by all four fungi, there were considerable differences in colonization of the other two plant species. Hedysarum coronarium showed the most striking differences, being colonized to the same extent as M. sativa by G. mosseae, but scarcely or not at all by G. caledonium or by one of the isolates of G. fasiculatum. Hedysarum coronarium is certainly not a ‘non-mycorrhizal’ plant, but there is clearly some degree of specificity in its response to different fungi, and this has not been further investigated. Fungal host range may occasionally also be restricted, for in a survey of 19 species of host plant, Glomus gerdemanni formed mycorrhizas only with Eupatorium odoratum (Graw et al., 1979). Genotype-dependent effects on the extent of colonization have also been observed in a number of species (see Smith et al., 1992; Peterson and Bradbury, 1995). In one recent example, the extent to which Glomus etunicatum colonized barley differed in different cultivars, and showed considerable cultivar-dependent response to P application (Baon et al., 1993), but care needs to be exercised in these comparisons because environmental conditions can influence the differences in colonization (Azcón and Ocampo, 1981; Vierheilig and Ocampo, 1991). The most extreme examples are the restriction of colonization at different stages in plant mutants or genotypes of otherwise highly mycorrhizal species such as Pisum sativum, Vicia sativa (Duc et al., 1989) and Medicago sativa (Bradbury et al., 1991). Investigation of these mutants may help to unravel the genetic control and physiological mechanisms determining mycorrhizal colonization. It is already clear that mycorrhizal colonization of roots can be blocked at a number of stages in typical host plants and that a number of different mechanisms are likely to be responsible for failure of colonization in non-mycorrhizal plants. We do not yet know enough about control mechanisms to say whether or not study of mutants will help with understanding those that prevent or restrict the colonization of naturally occurring non-host species by mycorrhizal fungi (see Chapter