The Physiology of Ectotrophic Mycorrhizas

The Physiology of Ectotrophic Mycorrhizas J.L. HARLEY AND D. H. LEWIS Department of Botany, The Uiaiversity of Shefield, England I. Intmductiori . 11. Carbohydrate Physiology . A. Carbohydrato Nutrition of Mycorrhizal Fungi B. Effects of Infection on Translocation and Photosynthesis C. Storage Polysaccharides in Infected Roots 1). Intensity of Infection and Soluble Carbohydrates . E. Mechanisms of Carbohydrate Transfer . 111. Fungal Mctabolites and their Effects on the Hosts . A. Morphogenesis of Mycorrhizas . B. The Ageing of Root Meristerns . C. The Effects of Hormones and Antibiotics of Fungal Origin IV. Host Metabolites and their Effects on the Fungi . A. Nutritional Requirements of Mycorrhizal Fungi in Culture B. Root Exudates and the Growth of Mycorrhizal Fungi . C. Defence Reactions of Host Tissue . V. Absorption of Nutrients by Ectotrophic Mycorrhizas . A. Mycorrhizal and Uninfected Roots as Absorbing Organs . B. Movement of Phosphate through the Fungal Sheath . C. Accumulation of Nutrients V1. Conclusions . References

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I. Introduction The term, mycorrhiza, has been applied to a wide variety of associations between fungi and other plants. I n an attempt to define the term precisely and so restrict its use, Harley (1959, 1968b) wrote, “In so far as associations of absorbing organs and fungi, constant in structure and development and consistently present and functional in natural conditions, are recognizable the name mycorrhiza may be legitimately applied to them”. The name was originally applied by Frank (1885) to the dual system present in the ultimate rootlets of members of the Cupuliferae (Fagales) and Pinaceae. In these species, the mycorrhizal condition is ectotrophic, that is, the fungal partner forms an enveloping sheath 53

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around the ultimate rootlets of the host, together with intercellular hyphae of limited extent, usually referred to as the Hartig Net. This type of infection contrasts with the endotrophic condition where there is no external sheath and intracellular penetration is the rule (see Harley, 1959,1968b; Mosse, 1963; Meyer, 1966; andNicolson, 1967for reviews of endotrophic associations). This article will be restricted to a consideration of the physiology of ectotrophic mycorrhizas, especially those of the European beech, Pagus sylvatica, and various pines, Pinus spp. A large number of fungi, mostly basidiomycetes of the Agaricales, Hymenogastrales and Sclerodermatales, are thought toformmycorrhizas (Trappe, 1962) but only about 100 have been shown to do so experimentally. The investigation of ectotrophic mycorrhiza has gone through a number of phases since the days of Frank. The first led to the acceptance of this infection as an essential feature of the absorbing organs of a large number of coniferous and angiospermous trees. During this phase, evidence was put forward that in some circumstances mycorrhizas had an absorbing efficiency greater than their uninfected counterparts. Later, the work of Stahl (1900) and also, from 1917 onwards, of Melin set the stage for further advance, so that in 1927 the extensive review of Rayner was able to show to botanists and to foresters that ectotrophic mycorrhizal infection was a feature of very many forest plants that required due consideration in the interpretation of their ecology and in their sylvicultural treatments. The investigations of Hatch (1937)on the genus Pinus laid a foundation for further physioIogica1 progress. He demonstrated, by field study and by laboratory work, two important characteristics. The first was that the intensity of infection in seedlings varied with nutrient supply so that mineral deficiency in low working ranges (but not in real starvation conditions) of nitrogen, phosphorus or potassium stimulated infection. Secondly, in conditions of limited supply, mycorrhizal infection improved the absorption and incorporation, per unit weight of pine seedlings, of compounds containing these elements. These findings were later amply confirmed by others especially Bjorkman (1942) who further elaborated Hatch’s views. The mechanism that Hatch propounded to explain the effectiveness of mycorrhizal organs in the uptake of nutrients from the soil was a physical one. He pointed out that, although the surface area of the root system was difficult to measure, there could be little doubt that the area of the absorbing surface of mycorrhizal seedlings exceeded non-mycorrhizal ones. Such an hypothesis would explain the effects of infection in stimulating the absorption of a number of nutrients rather than the single one, nitrogen, that had been mainly considered in some of the earlier hypotheses. The physical increase of root surface area was shown to be

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contingent upon an extension of the life of the axes of individualrootlets, an increase in the diameter of their host tissues, the presence of the fungal sheath and the existence of hyphal connexions between the mycorrhiza and the soil. This hypothesis of Hatch, derived as it was from a consideration of the earlier work of Stab1 and Melin as well as from much new experimentation, provided a basis from which many of the later ideas sprang. Subsequent work took note of the fact that, although the magnitude of the absorbing surface of mycorrhizal root systems was greater than with nonmycorrhizal ones, it was a different-a fungal-surface. Questions were therefore asked and answers were sought experimentally about the absorpt,ive properties of this surface and about the intervention of the fungal layer in the processes of uptake. I n the meantime, from the 192O’s, Melin with his pupils and colleagues had been considering the fungal partners of ectotrophic mycorrhizal symbiosis and it had become apparent that they were mainly basidiomycetes. Besides requirements for trace substances, known vitamins, metabolic intermediates, and root exudates, Melin and his colleagues showed them to require as a general rule simple soluble carbohydrates for growth (see Melin, 1963). It was therefore reasonably surmized that these special requirements, including carbohydrates, were obtained from the host. Bjiirkman, from 1942 onwards, brought together these properties of the fungi and the results of Hatch to propound and test a further hypothesis concerning the conditions for infection and the development of mycorrhizal organs. I n his experiments with conifer seedlings, the degree of development of mycorrhizal infection was dependant on light supply. Bjiirkman tested the proposition that the effect of light was through its effect on photosynthesis and so upon the supply of simple carbohydrates to the root and hence to the fungus, by analyses of seedlings growing in artificial or natural soils under different conditions of light, and nutrient supply. He observed a correlation between the intensity of infection and the quantity of easily soluble reducing substances in the root systems. The effects upon infection of differing amounts of nitrogen-, potassiumand phosphorus-containing nutrients supplied under a single light regime were interpreted through changes in soluble reducing substances. It was held that nutrients which allowed growth and the utilization of carbohydrate decreased the concentrations of these compounds in the root system and so diminished mycorrhizal infection. Bjorkman’s hypothesis, unifying as it did many previous lines of work, was a powerful influence on subsequent thinking and experimentation. I n the course of time, as shown below, other lines of work and a re-examination of the earlier experimental results have suggested that a modification or extension of it may be necessary.

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Hatch in his consideration of the area of mycorrhizal root systems had clearly summarized the differences between those that were mycorrhizal and those that were not. Amongst his points, some have proved to be linchpins for the wheels of later advance. The first relates to the longevity of mycorrhizal axes. Hatch and Doak (1933) pointed out that the ultimate rootlets of pine were of three kinds. The first kind were uninfected short roots which might continue slow growth and dichotomize "frequently but not profusely". These might be attacked by fungi and converted into the second kind, pseudomycorrhizas, which then ceased growth. The third kind were mycorrhizal short roots which continued to grow and dichotomize for a long period of time, and were the absorbing organs of the system. This difference of behaviour of mycorrhizal rootlets from others has since been shown to be general in conifer and angiosperm alike, with the result that the suggestion has been put forward that one effect of infection is to prolong the life of tissues of short roots. A second cardinal point of Hatch concerns the increase in diameter of infected host tissues consequent upon the characteristic radial elongation of epidermal and cortical cells of the root in mycorrhizas. These features of the dual system have led to questions of mechanism and so to investigations of the production of auxins and other growth substances by mycorrhizal fungi and their possible role both in growth promotion and longevity of the root and in the modification of its morphology and anatomy. Slankis (1948 and later references) was the initiator of this productive line of work. The slow growth of mycorrhizal fungi in culture, even in the presence of suitable substrates and trace amendments, contrasts with their apparently ready spread in the root region of susceptible hosts. This kind of consideration led Melin and his school to examine host metabolites that, by being released or by existing in the tissues in sufficient quantity, might stimulate fungal growth (Melin, 1963). The discovery of such substances has led to work on factors which might alter their production or availability. Little activity, other than thought, has been given to similar factors which could alter the form of fungal growth from mycelium to pseudoparenchyma, and so explain the formation of the external fungal mantle or sheath. It is obvious that these facets of investigation on mycorrhizal physiology have come in contact with those on rhizosphere effects and especially with those on root secretions and their variations. In this review, the subjects to be assessed will be solely those contributing to the physiological understanding of ectotrophic mycorrhizas. Firstly will be considered three aspects of the establishment and maintenance of the characteristic dual organism, namely carbohydrate metabolism, fungal metabolites and their effect on the hosts and host metabolites and their effect on the fungi, Secondly, the process of nutrient

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absorption will be appraised for this is assumed to be the main ecological role of the fungi. Progress in these investigations has not been rapid, and the function of this review is to integrate observations on mycorrhizas into the wider context of plant pathology and plant physiology and to highlight problems rather than to present final answers. On some issues, speculative lines have been adopted, but we feel that any hypotheses presented are capable of being experimentally tested by a combination of available techniques.

11. Carbohydrate Physiology

A. CARBOHYDRATE NUTRITION OF MYCORRHIZAL FUNGI The work of Melin and his colleagues (see Melin, 1963) on mycorrhizal fungi in culture emphasized their requirement for simple carbohydrates, arid this reinforced the supposition that, in nature, they depend upon photosynthetically produced carbohydrates from their hosts. The observation of Rommell (1938) that several species failed t o produce fruitbodies when isolated from the roots of host-plants also supports this view. It has frequently been observed that mycorrhizas develop on seedlings only in conditions where an adequate rate of photosynthesis is possible. The roots of seedlings of diverse species of tree become mycorrhizal only after the first foliage leaves have expanded (e.g. Huberman, 1940; Warren-Wilson, 1951 ;Robertson, 1954; Boullard, 1960,1961 ; Laiho and Mikola, 1964). I n addition, an adequate light supply must be available as has been shown either by varying light intensity or duration of illumination (Bjorkman, 1942; Wenger, 1955; Harley and Waid, 1955; Hacskaylo and Snow, 1959, Boullard, 1961). Some estimate of the magnitude of the movement of carbohydrate involved in the nutrition of the mycorrhizal fungus, supposing it to be totally dependant on its host for carbon-containing nutrients, may be obtained by examining the results of dissecting the apical tips of beech mycorrhizas. Harley and McCready (1952) showed that the fungal sheath comprised 39% of the total dry weight of the mycorrhizal axis. From this it would follow, allowing for respiration, that a t least 40% of the products of photosynthesis passing to the feeding roots would be consumed by their fungi. It is of interest in this regard that Tranquillini (1959, 1964) arrived a t a similar figure by studying carbon exchange in Pinus cembru in the field. It follows from these considerations that, if the fungus is nourished entirely by the host, it must be a niajor drain on carbohydrates translocated to roots. Evidence of this kind does not demonstrate unequivocally that there is a movement of products of photosynthesis into the fungus. but this

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has been shown by experiments with radioactive carbon dioxide. I n 1957, Melin and Nilsson supplied the shoots of normal and decapitated seedlings of Pinus sylvestris with 14C02in the light. The seedlings had been raised in two-membered culture with either Boletus variegatus or Rhixopogon roseolus, and had developed mycorrhizas. Fixed carbon compounds were found in the mycorrhizas of intact seedlings in excess of that in decapitated seedlings. Dissection of apices demonstrated that radioactivity had passed to fungal tissue. I n field experiments performed for a different purpose, Bjorkman (1960) injected labelled glucose into spruce and pine, and also showed translocation of carbon compounds from the host to the mycorrhizal fungus. Something of the rate and mechanism of movement of carbohydrate was learned by Lewis and Harley (1965~)in their work with excised mycorrhizas of beech, Fagus sylvatica. They applied 14C-sucroseto the cut stumps of mycorrhizal tips and found that the sugar was translocated through the host tissue to the tip. The apical 5 mm. were excised and dissected to remove the fungal sheath after periods of 22-27 hours. Between 55 and 76% of the 14C which had passed to and remained in the tip was in the fungal layer. Within host tissue of the core, sucrose remained heavily labelled but, in the sheath, the radioactivity was largely distributed between the fungal carbohydrates, trehalose, mannitol and glycogen. Both mycorrhizal and uninfected roots can absorb and utilize glucose, fructose and sucrose (Lewis and Harley 1965a, b), but the host tissue is virtually unable to absorb or utilize trehalose and mannitol, which the fungus can utilize readily. The fungus can therefore be viewed as a metabolic sink which receives carbohydrate from the host and converts these into forms which cannot be utilized by the host. There is no reciprocal flow. This mechanism appears to be common to many mutualistic and parasitic organisms which derive carbohydrate from their hosts, e.g. the fungi of lichens, ecologically obligate associations of fungi and algae, biotrophic parasitic fungi of higher plants, and some parasitic angiosperms (Smith et al., 1969). Of special significance here is the similarity of the transfer process from barley to the powdery mildew, Erysiphe graminis, studied by Edwards and Allen (1966), for this parasite has a n almost ectotrophic nutrition. B. EFFECTSOF INFECTION ON TRANSLOCATION AND PHOTOSYNTHESIS The fact that carbohydrate is diverted irreversibly to the fungus might be expected to have repercussions on patterns of translocation within the host plant. This has not been investigated for the European species of pine, spruce and beech but has been studied for Pinus resinosa and P. strobus in Canada (Shiroya et al., 1962; Nelson, 1964; Lister et al., 1968).

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Mycorrhizal root systems always accumulated more photosynthetically fixed 14C per unit time than non-mycorrhizal roots. Similar changes in the pattern of translocation occur in plants infected by pathogenic fungi, a feature of disease also reviewed by Smith et nl. (1969). The Canadian workers also determined the pattern of labelling in carbohydrates and other soluble compounds in the root systems of pine, and always found sucrose to be most heavily labelled irrespective of experimental treatment (mycorrhizas present or absent ; growth of seedlings under different mineral conditions). I n heavily mycorrhizal roots, it seems odd at first sight that fungal carbohydrates such as trehalose and mannitol were not recorded as radioactive. This can be explained simply by the fact that, in the whole root systems analysed, the host tissue was quantitatively very large compared with the fungal tissue with the result that fungal metabolites may have easily been undetected. These observations of increased translocation to mycorrhizal root systems led Harley (196th) to speculate on the same lines as Thrower (1966) that such fungal infection may indirectly increase the rate of photosynthesis. From studies on photosynthesis in rooted detached leaves of bean, and from an appraisal of earlier data, Humphries (1963) concluded that the rate of photosynthesis was governed by the rate of translocation to a sink. Sweet and Wareing (1966) also consider their data on photosynthetic rate and growth in Pinus mdiutn to be consistent with this hypothesis, and further that movement of products of photosynthesis is under hormonal control. That mycorrhizal infection may increase the rate of photosynthesis is supported by the observations of Lister et ul. (1968) whose tables of data show that the highest rate of net photosynthesis is correlated with the highest degree of mycorrhizal infection. An increase in the rate of photosynthesis is a feature of the early stages of infection by several fungal pathogens of leaves (Yarwood, 1967). This aspect of the mycorrhizal plant where infection remains benign for a long period requires further study for it is potentially of great ecological and economic significance.

C. STORAGE POLYSACCHARIDES IN INFECTED ROOTS A common feature of many infections of leaves by biotrophic fungi is the accumulation of starch around the pustules within host cells (Thrower, 1965), i.e. in an affected but uninfected zone. In view of the parallels between such infections and those of roots by mycorrhizal fungi noted above, it is significant that Harley and Jennings (1958) recorded the presence of starch, particularly in the pericycle, in beech mycorrhizas. In a recent study of mycorrhizal roots of Pinus radiatn by electron microscopy, Foster and Marks (1966) showed that cells of the cortex not

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infected by fungus contained amyloplasts filled with starch, whereas COPtical cells in contact with fungus of the Hartig Net had empty amyloplasts. The cells containing starch may be comparable with the affected zone of infections of leaves. Foster and Marks (1966) also demonstrated a massive accumulation of glycogen in fungal hyphae of the Hartig Net with a lesser quantity in the sheath, recalling the earlier studies of Rexhausen (1920). That the Hartig Net contains a higher concentration of glycogcn than the sheath provides a possible explanation for an observation of Lewis and Harley (196513).They found that 20yo of the incorporation of I‘C-glucose into glycogen by excised mycorrhizas was found in the core, i.e. in fungus of the Hartig Net. If glycogen was evenly distributed. this is an unexpected result since the dry weight of the sheath must be more than four-fold greater than that of the Hartig Het. This relatively greater incorporation into the latter structure is explicable in terms of the distribution of glycogen if beech mycorrhizas have the same pattern as p inc,

D. INTENSITY OF INFECTION AND SOLUBLE Although it appears probable that both the host and fungal tissues of mycorrhizas receive a large part, perhaps all, of their carbon from photosynthesis, it does not follow that there should be a correlation between thc amount of soluble carbohydrates in the root system and the intensity of mycorrhizal infection. A s noted above, the hypothesis that there was such a causative correlation was first put forward by Bjorkman (1942). Some othcr workers have obtained results in agreement with Bjiirkman (e.g. Harley and Waid, 1955) but many have not (e.g. Warren-Wilson, 1951; Meyer, 1962). Richards and Wilson (1963) and Richards (1965), while not finding a correlation of infection with carbohydrate content, (lid obtain one between infection and the ratio between the carbohydrate and nitrogen contents of the root system. I n all this work, there are two possible sources of error, one in the experimental design used by Bjorkman and those that followed him and a second in the methods of analysis used for carbohydrate assay. Handley and Sanders (1962) pointed out that, in the experiments of Bjorkman, the root systems analysed had variable amounts of mycorrhizal development on them. It was therefore arguable that the different concentrations of easily soluble reducing substances which were found in I oot systems with different mycorrhizal development arose from the fact that the fungus contained, or caused the mycorrhizas t o contain, larger amounts of these compounds per unit weight than uninfected tissue. They therefore repeated Bjorkman’s experiments as exactly as possible except that no mycorrhizal fungus was present and no mycorrhizas were

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formed. They observed that the internal concentration of reducing substances in the roots did not increase with increasing light intensity as they did in Bjorkman's experiment. Hence they concluded that mycorrhizal infection was the cause rather than the resultant of the different carbohydrate concentrations which Bjorkman observed. Meyer (1962, 1964, 1966) examined the effects of light intensity and nutrient supplies on mycorrhizal infection of beech and spruce in soils of different nutrient status. After failing to obtain the kinds of correlation between infection and carbohydrate status that would be expected of Bjorkman's hypothesis hc concluded, like Handley and Sanders (1962),that the variations in sugar content of the root system were more likely to be a resultant of fungal infection than an antecedent cause. I n this he agreed with the work of Warren-Wilson (1951) who, using beech, concluded that there was no general correlation of sugar content of the roots and mycorrhizal development. These experiments throw doubt, not upoii the conclusion that myrorrhizal fungi depend for some or all of their carbon compounds on their hosts, but upon the hypothesis that there is it correlation between the carbohydrate content of roots and intensity of infection. The second cause of doubt arises from a consideration of analytical methods. Lewis (1963) pointed out that the various analytical procedures used by different people estimated different carbohydrate fractions and variakde amounts of non-sugar reducing compounds. Bjorkman was indeed aware of this and referred t o easily soluble reducing substances rather than soluble carbohydrates. Nevertheless it was implied that the estimates of reducing power obtained were a t least rough estimates of carbohydrate. Later workers have used other methods of extraction, clearing and estimation more likely to give more accurate determinations of carbohydrate, but none has been quite satisfactory. Lewis and Harley (1965a) showed that only 50% of the reducing power extracted by 8076 ethanol from beech mycorrhizas was due to reducing sugars. The other reducing substances were charged and neutral compounds which could bc removed by deionizing and clearing. Amongst the substances removed in this way were glycosidic cornpounds which would be estimated as carbohydrate by the anthrone procedure but not by other methods. I n addition, the mycorrhizas contain the non-reducing disaccharides sucrose and trehalose and the hexahydric alcohol, mannitol. Of these compounds, sucrose might have been readily hydrolysed and estimated as reducing sugars but trehalose is resistant to hydrolysis by dilute acid and mannitol cannot be estimated a t all by the methods used. The hydrolysis of sucrose and trehalose may be brought about inadvertently in some extraction procedures. For instance, if the material is preserved or killed by drying a t 80" or less before extraction by water, the enzymes invertase and trehalase which it contains may not be denatured. Hydrolysis

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of sucrose or trehalose may proceed during subsequent extraction so that the estimates of free reducing sugar in the tissue are faulty. As a result of these considerations, although it must be accepted that tfhe fungus receives carbon compounds from the host and that these are derived from photosynthesis fairly directly, no good evidence is available that the degree of development of infection and the soluble carbohydrate content of the roots are quantitatively correlated. It is more relevant to an understanding of the physiology of mycorrhiza to consider further the mechanism of carbohydrate transfer between the symbionts.

E. MECHANISMSOF CARBOHYDRATE TRANSFER This whole process may, for convenience, be divided into five stages : (a) stimulation of movement of carbohydrates to the affected and infected areas ; (b) stimulation of polysaccharide synthesis in non-infected cells of the affected area; (c) release from affected cells of soluble sugars derived either directly from the main translocation stream or from this polysaccharide store; (d) absorption of sugars by the fungus; ( e )conversion of absorbed sugars to fungal storage products. Whereas little experimental data directly concerned with the first three facets are available for mycorrhizas, relevant investigations concerning the two latter have been conducted. The mechanism of absorption of carbohydrates by the mycorrhizal fungus of beech has been studied by Harley and Jennings (1958) by use of excised mycorrhizal roots. Continued absorption of the monosaccharides, glucose and fructose, occurs via a metabolically dependent process in that it is sensitive to external pH values, and to inhibitors such as 2,4-dinitrophenol and silver nitrate. It responds to increased concentration of hexose in a typically hyperbolic manner. Glucose is preferentially selected from mixtures of the two hexoses. From a study of the factors affecting the absorption of sucrose, they concluded that this disaccharide was first hydrolysed to its constituent hexoses from which glucose was again preferentially selected. These characteristics are found in many free-living fungi, lichens and higher plant tissues but others appear to absorb sucrose intact (see Hardy and Norton, 1968). Lewis and Harley (1965a, b, c) investigated the fate of sugars absorbed by excised mycorrhizas both from solution and in a system designed to simulatenatural translocationto theroot apices. Glucosemoieties, derived either from free glucose or sucrose, were principally converted to trehalose and glycogen whereas fructose moieties, also either from free fructose or sucrose, were converted to mannitol. Similar behaviour, reviewed by Smith et al. (1969), has been noted in many associations involving fungal pathogens. Biochemical mechanisms involved in this differing

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fate of the two hexoses are not understood, but the first speculation of Lewis and Harley (1965b) that the mycorrhizal fungus had low activity of glucose phosphate isomerase (phosphohexoisomerase) proved unfounded (Harley and Loughman, 1966). As well as the need for further investigation on the absorption and metabolism of host carbohydrates by mycorrhizal fungi-it must be emphasized that all generalizations are a t present derived from observations on beech-we are almost totally ignorant of the manner by which the fungus alters the physiology of the host to bring about the first three facets of the diversion of host photosynthetic products to its own ends. Fungal metabolites are undoubtedly involved and are discussed in the next section.

111. Fungal Metabolites and Their Effects on the Hosts The tissues of ectotrophic mycorrhizas, both host and fungal, differ in structure from those of the constituent organisms in culture or in the free state. I n this they resemble those of other parasitic and mutualhtic systems such as galls, lichens and bacterial nodules. I n addition to these morphological and anatomical differences, physiological interactions also occur, e.g. those mentioned above resulting in the diversion and leakage of photosynthetic products of the host to the fungi. To explain these, hypotheses have been put forward from time to time that metabolites of each organism are responsible.

A. MORPHOGENESIS OF MYCORRHIZAS The characteristics of the host tissues of ectotrophic mycorrhizas have been attributed over the years to morphogenic stimuli produced by the fungi; at first with no experimental backing and recently with more credibility following the discovery of auxin-like fungal metabolites. But there is one early step in the argument which, although fundamental, is as yet not completely resolved. It is not fully known to what extent the characteristics of the host tissues, which depart in structure from rapidly growing uninfected roots, result from the activities of the fungus and to what extent they depend upon normal processes of development of the host. These differences have been aptly summarized by Clowes (1949, 1951) for Fccgus sylvaticcc and by Chilvers and Pryor (1965) for various species of Eucalyptus, and their accounts are generally applicable. Mycorrhizal axes exhibit differentiation of the stele, cortex and endodermis nearer to the meristematic apex than uninfected roots. This is usually associated with an obliquely lateral elongation of the cells of the outer cortex and epidermis and a reduction of their length. The apical

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meristem is similar in architecture to that of uninfected roots, and the sequence of divisions is also similar. There is evidence, considering for instance the ratio of stelar to cortical diameters, that the diameter of infcctcd is greater than that of uninfected axes and that the increase arises from the lateral expansion of the cortex in particular. Except in rare instances, such as in the superficial ectotrophic mycorrhizas of Fagus, no root hairs are produced. I n certain roots, a layer of cells, the walls of which are impregnated with tannin, is present in the cortex of mycorrhizas and through it the fungus only penetrates with difficulty. Although most people have tacitly assumed that these differences were due to fungal activity, this is by no means certain. Hatch and Doak ( 1933) and Hatch (1937) pointed out that the short rootlets of Pinus soon abort if uninfeeted and that mycorrhizal infection promotes their growth, branching and longevity, and this has been the interpretation of many later observers. Warren-Wilson (1951) studied the root systems of Pagus seedlings growing in natural soils from the time of germination until the end of their first year. He observed that before fungal infection occurred the tips of many of the branch rootlets underwent a change so that they approximated in structure some of the characteristics of mycorrhizasnamely in differentiation of tissues close t o the apex. It was after this change that mycorrhizal infection occurred. I n the latter months of this first year, the proportion of normal and aged uninfected root tips greatly diminished and the surviving rootlets were dominantly mycorrhizal. Warren- Wilson (1951) obtained some evidence that this process of ageing of the apices might occur in the absence of fungi. Moreover Clowes, although of the opinion that the fungus caused change in the host, observed that a Hartig Net was only found between cells which were already laterally expanded. I n this context the observations of Baylis et al. (1RB3), Becking (1965) and Khan (1967) on nodules of Podocarpus are relevant. The nodules, which under natural conditions harbour a fungal endophyte, are not dependent on the fungus for development, for they form on the roots of aseptically grown embryos. The possibility therefore exists that two sets of morphogenic changes co-operate to produce the mature structure of the host tissue in mycorrhizal axes-an endogenous ageing process which arises from the host and a maintenance of growth and branching due to fungal activity.

B. THEAGEINGOF ROOTMERISTEMS The process of the ageing of the meristems of roots has been recently reviewed by Street (1967). Most of the information relates to work with excised roots in culture and, as Street emphasized, very little work has been done on the behaviour of the apices of roots of whole plants. I n

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brief', the facts are that some roots, tomato and Senecio have been much used, undergo a progressive ageing of their meristems. With time, or on continued subculturing of the apex, growth ceases often after a nearly constant amount of tissue has been formed. The processes are hastened by high exogenous sucrose concentrations so that there is a critical sugar concentration a t which growth is possible, yet a t which a minimum of ageing in terms of survival or growth occurs. The presence of exogenous P-indolylacetic acid (IAA) increased ageing. I n some cases roots in high conc.ent,rations of sucrose (3-40/,)exhibited reduction of linear growth rate, increased root diameter, and increased frequency of laterals, in a manner similar to that of roots in lower ( 2 % ) concentrations of sucrose with exogenous IAA. The similarity of roots inhibited by sucrose and IAA rxtended to anatomical and histological features including radial increase of the cortical cells. These kinds of results have suggested that a auxin-like regulator or ageing factor may accumulate in supra-optimal amounts in the mcristem and eventually lead to ageing. Further tests with pure auxins showed that naphthalene acetic acid (NAA) a t low concentrations not only has similar effects but could k i n g about progressive or cumulative ageing of the meristem. For instance a concentration of 1 0 - ''I g. NAA per ml. was without effect on tomato roots in the first passage in a culture medium containing 2% sucrose, but, on repeated subculture in even lower sucrose concentrations, it brought about progressive ageing of the meristems. Naphthalene acetic acid therefore mimicked closely the action of the postulated ageing factor in its cumulative effect. With roots ofsenecio, 2-naphthoxyacetic acid seems to play a similar role in simulating the ageing factor, and once again sucrose concentration interacts with and modifies its effects. A further complication is the existence of anti-ageing factors such as 1-naphthoxyacetic acid, which appears to prevent or remove the ageing effect of NAA but not of IAA. Gibberellic acid can in some circumstances promote ageing whereas kinetin, in these assays, is an anti-ageing factor capable of antagonizing the effects of NAA and gibberellic acid. Street (1967) concludes that rncristematic activity appears to be controlled by the relative endogenous levels of an auxin and a kinin and is also dependent on carbohydrate SUYP'Y. This work is of particular interest in the light of observations made by Clowes (1949, 1952) and Chilvers and Pryor (1965). Clowes showed that ZAA applied in lanolin to the root apices of Fagus seedlings caused decreased growth and local swellings in which the radial and tangential sizes of the cortical cells were increased, as well as a proliferation of lateral primordia. Roots of seedlings grown in O-Olyo(w/v) solution of colckicine were also modified to resemble mycorrhizas by having no alteration of the architectural pattern of their apices, whilst exhibiting

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vacuolation and maturation very close to the promeristem and transverse enlargement and reduction in length of the cortical cells. Chilvers and Pryor (1965) applied NAA to the roots of Eucalyptus grandis growing in sand and perlite. High concentrations inhibited root growth and branch initiation completely and low concentrations ( 10-6-10-7 M) stimulated linear growth. Concentrations of 10-5-10-6 M decreased linear growth, increased the diameter of the growing apices by 70-100%, and caused the initiation of short side-branches. This produced an uninfected system externally resembling mycorrhizal infection except that large main roots were also modified. Anatomical investigation of these systems showed that differentiation occurred close to the apices but, although the epidermal cells were slightly increased in radial and tangential diameters and reduced in axial diameter, the changes fall short of those seen in mycorrhizal roots. Nor was there any thickening in the walls of the cortical cells. The volumes of the cortical cells were increased four- to five-fold, an hypertrophy unlike anything seen in mycorrhizal roots. They also report that colchicine caused distortion of the root-tips due to combined increases in diameter of epidermis, cortex and stele. Chilvers and Pryor (1965) were of the opinion that such results as these were merely a pointer to the manner in which further advances might be made, because the anatomical and histological changes only partially matched the effects of mycorrhizal infection. They pointed out that factors which affect growth of the root axis result also in development of some of the anatomical characteristics of the host tissue of mycorrhizal roots ; “Ageing roots, roots growing slowly through unfavourable media, and roots artificially restrained by overdoses of colchicine and naphthalene acetic acid all exhibited the same phenomenon”. C. THE EFFECTSOF HORMONES AND ANTIBIOTICS OF FUNGAL ORIGIK The knowledge that mycorrhizal basidiomycetes, notably Boletus edulis, could produce auxins or auxin-like substances in culture solutions is of long-standing. I n a series of papers (1948, 1949, 1951), Slankis demonstrated that culture filtrates of Boletus variegatus and other boleti could bring about dichotomy in short roots of pine. I n the later papers, he showed that IAA, as well as other indolyl compounds and NAA in suitable concentrations, were also active in this regard. They caused stimulation of growth a t low concentrations and inhibited the growth of short axes a t higher concentrations whilst stimulating dichotomous branching. Naphthalene acetic acid was more active in these ways than IAA. Since then the production of indolyl compounds in culture, especially from tryptophan, has been shown to be a general property of many mycorrhizal fungi (e.g., Moser, 1959; Ulrich, 1960a; Horak, 1964) and it

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has been assumed to be of first importance in determining the morphology and anatomy of the host axes of mycorrhizal organs. Nevertheless Ulrich (1960b) was unable to show that PAA reproduced mycorrhiza-like modifications in roots of Pinus lambertiana in culture, and Barnes and Naylor (1959a, b) obtained dichotomy in Pinus serotina using a variety of compounds in culture solutions. The later work of Slankis (1955,1961, 1963) is of especial relevance. He has not only been able to stimulate in aseptic conditions the production of dichotomous systems similar to coralloid bunches of mycorrhizas or tuberculate mycorrhizal systems in culture with appropriate IAA concentrations, but has also published photographs showingthat histological patterns similar to those of mycorrhizas may be produced. Slankis (1958) also showed that exogenous auxin not only affected the short roots directly but was absorbed and translocated throughout the root system. It could therefore affect growth and production of short roots. More interesting still was the reported correlation of auxin effects and photosynthesis. Slankis (1961,1963)reported that, at low light intensities such as led to little mycorrhizal production in Bjorkman’s experiments (1942), auxins had only slight morphological effects on pine roots even at high concentration. At higher light intensities or in the presence of adequate concentrations of sugar they caused the expected alterations of form and structure. This result is greatly reminiscent of the work discussed by Street (1967) for excised roots of tomato andSenecio in culture. Similarly, Wetmore and Rier (1963) have shown that the experimental induction of vascular tissues in callus cultures of several angiosperms is dependent upon an interaction between auxins and sugars, the concentration of the latter being of prime importance in determining the ratio of xylem to phloem. A further interesting line of speculation arises from an observation by Moser (1959) that auxin production by mycorrhizal fungi in culture was diminished when high concentrations of nitrogen-containing compounds were available. This of course is reminiscent of the results of Hatch, Bjorkman and others that adequate nitrogen supply in the soil tends to decrease mycorrhizal production. Although none of this work answers the question unequivocably whether ageing of root apices precedes or follows fungal infection, it does indicate that photosynthetic rate and sugar production in the host, together with mineral supply in the soil, may indeed be important together with the production of morphogenic substances by the host, the fungus or both, in the interaction of ectotrophic mycorrhizal infections. Since growth processes in plants involve the interaction of several classes of hormone including gibberellins and kinins as well as auxins, the 3

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role of these compounds in the morphogenetic changes in roots consequent upon infection therefore merits consideration. Although gibberellins were first isolated as products of fungal metabolism, the growth of several mycorrhizal basidiomycetes but not Cenoccocum graniforme appears to be severely limited by gibberellic acid (Levisohn, 1960; Santoro and Casida, 1962). It therefore seems unlikely that this particular class of hormone is a significant fungal product important in the establishment of the mycorrhizal condition. Kinins, on the other hand, are very likely to be involved. Arora et al. (1959) noted that kinetin induces, on the roots of tobacco in culture, the formation of pseudonodules which develop by division and enlargement of cortical cells. I n view of this enlargement which is similar to that in mycorrhizal roots, Miller (1967) examined culture filtrates of Rhizopogon roseolus, a known mycorrhizal fungus, and isolated zeatin, zeatin riboside and at least one further kinin. The importance of this observation is obvious, but has yet to be exploited experimentalIy by students of the morphogenesis of mycorrhiza. Similarly the effects of fungally produced hormones on physiological and metabolic changes of mycorrhizal host plants have not been investigated, but from comparisons with other studies they are likely to be profound. Evidence is now accumulating that patterns of translocation in higher plants are under hormonal control (Letham, 1967 ;Seth and Wareing, 1967). It therefore seems probable that the changes of this nature consequent upon mycorrhizal infection are mediated by fungal production of plant hormones. This aspect of fungal infection has been investigated experimentally for rust infections by Pozsk and Kirsly (1966) and Kir&lyet al. (1967) and reviewed in a wider context by Smith et al. (1969). It is also possible that hormones, especially auxins, may be responsible for the hydrolysis of starch in the affected cells (Meyer, 1966). Since auxins are also known to increase the permeability of plant cells, they may mediate the leakage of carbohydrate from cortical and epidermal cells to the fungal hyphae of the Hartig Net and sheath. A further manner by which mycorrhizal fungi may affect the growth and metabolism of their hosts is through the production of antibiotics. These compounds may act directly on host cells, e.g. by altering cell wall metabolism or cell permeability as suggested by Smith et al. (1969) or indirectly via their protective action in preventing pathogenic infection (Zak, 1964; Ohara and Hamada, 1967; Marx and Davey, 1967). Zak’s (1964) review gives earlier references to the production of antibiotic substances by mycorrhizal fungi, am aspect of their metabolism more recently investigated by f$agek and Musilek (1967, 1968).

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IV. Host Metabolites and their Effects on the Fungi Harley (1 948) pointed out that ectotrophic mycorrhizal fungi should be viewed as specializedfungi of the rhizosphere which become dominant on the root surface. Within this specialized habitat the fungi grow as a weft of pseudoparenchyma closely adpressed to the root surface. To effect such development the fungi have, as it were, to know two things-where to grow and how to grow. As noted in the Introduction (p. 56), total ignorance exists concerning the latter but two, initially distinct, avenues of research have a bearing on the former; the determination of firstly the nutritional requirements for germination and growth of mycorrhizal fungi in culture and, secondly, the nature of exudates from roots in the rhizosphere.

A. NUTRITIONAL REQUIREMENTS OF MYCORRHIZAL FUNGI IN CULTURE I n contrast to the spores of most Iignin-and cellulose-destroying basidiomycetes,the spores of mycorrhizal species are notoriously difficult to germinate in the laboratory. They are frequently characterized by a low respiratory rate which Kneebone (see Benedict et al., 1967) suggested was due not to a lack of metabolic reserves, but rather some deficiency which prevents efficient utilization of available respiratory substrates. This deficiency may be supplied by other micro-organisms as Fries (1941, 1943) showed by the fact that germination could be obtained in culture when spores were plated with certain non-spreading microorganisms, e.g. Torulopsis sanguinea. Exudates of an unknown nature from roots of pine or tomato also stimulate the germination of spores of many mycorrhizal fungi (Melin, 1962). These compounds, collectively termed the M-factor, are discussed more fully below in connection with mycelial growth. However, Benedict et al. (1967) were unable to obtain spore germination of 13 species of basidiomycete by addition of either exudates from tomato roots or NAD, a compound which Melin (1 963) noted could replace the M-factor. Most data on mycelial growth of mycorrhizal fungi derive from the work of Melin and his co-workers. The fungi characteristically rely on simple carbohydrates and are commonly heterotrophic for various vitamins, amino acids and other known growth-promoting substances. I n addition, as elaborated below, many species, notably Russula xerampelina, Cortinarius glaucopus and Pholiota caperata, are especially fastidious and require unidentified compounds or the presence of roots themselves in order to grow (see Melin, 1963). An exudation of sugars and related compounds from roots would result in a non-specific stirnubtion of fungi such that mycorrhizal fungi with a low competitive saprophytic

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ability would be overgrown. Considerations such as these have led to the search for other, more specific, stimulatory factors.

B. ROOTEXUDATES AND THE GROWTH OF MYCORRHIZAL FUNGI The nature and effects of root exudates have been reviewed by Schroth and Hildebrand (1964) and Rovira (1965). Rovira gives tables of the kinds and quantities of carbohydrates, amino acids, organic acids, vitamins and a multiplicity of other compounds found in the root region of plants. There is a considerable variation in kind and in quantity according to species and the age, stage and conditions of growth. His own work (Rovira, 1959)shows that high light intensity increases the exudation of amino acids and might be expected to affect that of other substances. Slankis (1958) and Slankis et al. (1964) demonstrated the appearance of photosynthetically fixed carbon in root exudates of Pinus strobus. Sterile seedlings were exposed to 14C02for 8 days with a 16-hour photoperiod, and root exudates analysed chromatographically and electrophoretically. The identity of compounds was in some cases confirmed by the co-chromatography of derivatives. The major radioactive compound was malonic acid, but 35 other compounds also became labelled including other organic acids such as malate, glycollate, shikimate, oxalate and cisaconitate. The sugars, glucose and arabinose, and the amides, asparagine and glutamine, but not their corresponding amino acids, also became labelled. It may be that one or more of these compounds or some combination of them is important in stimulating mycorrhizal development, but it is more likely to be an unusual compound rather than a typical metabolite playing a major role in the normal metabolism of plants. The examination of exudates of sterile roots or root systems is only a first stage in a difficult search. Not only have changes in exudation rate and kind with cultural conditions to be examined but also the effects of the populations of the rhizosphere on the exuded compounds. As has been noted above, mycorrhizal fungi grow slowly in culture. This behaviour contrasts with their apparently rapid colonization of the roots of their hosts, a phenomenon which has led to investigations of their special nutritive requirements. Melin as long ago as 1925 first suggested that secretions from host tissues were necessary for growth of mycorrhizal fungi, an hypothesis amply confirmed by subsequent experimentation (see Melin, 1963). The unknown stimulatory compounds, caIIed the Mfactor, were not produced by susceptible hosts alone, but also by other plants including some herbaceous angiosperms which do not produce ectotrophic mycorrhizas. By placing living roots either directly in the cultures of the fungi or separating them from the media in celluloid bags,

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it was shown that diffusible substances which caused increased growth were produced. Some mycorrhizal fungi were stimulated only a little and required the M-factor solely in the early stages of growth. Others grow very poorly without the factor and were totally dependant on its presence throughout the growth period used (80 days). Melin used an arbitrary standard unit of the M-factor for quantitative work, namely the amount of active substance diffusing into water at 4' in six days from a quantity of living root equivalent to 1mg. dry weight. Using Boletus variegatus, he showed that low doses caused stimulation whereas high doses inhibited growth. From this and other experiments it was concluded that the exudate contained both stimulating and inhibitory substances, although it was not conclusively demonstrated that the M-factor's effect varied with concentration from a promotion of growth at low concentrations to an inhibition at higher values. Both the inhibitory and stimulating principles were differently distributed in the exudate from different parts of the root system of Pinus sylvestris. The old secondarily thickened axes produced an excess of inhibitory substances and the primary rootlets an excess of stimulatory ones. Melin (1963)has also reviewed the evidence in favour of a non-diffusible M-factor. Growth of Boletw variegatus in six days is almost doubled by the addition of diffusible M-factor (from one root for six days) but, if roots from which exudate was obtained are added either live or heatkilled, a further stimulation to almost three times the control is obtained. Since a marked response was elicited by the dead roots in the absence of the diffusible stimulator, the presence of a non-diffusible factor was inferred. It seems possible that this may be a thigmotropic response, a common feature of fungi. The response of mycorrhizal fungi to inert materials, e.g. nylon, of the dimensions of the root requires investigation. As yet, the chemical nature of the components of M-factor is unknown. Melin (1959)showed that they are prevented from being effective by the addition of adenine and some of its derivatives, although he also reported (Melin, 1963) that Nilsson had some evidence that the diffusible factor could be replaced by NAD. Benedict et al. (1967)found that NAD was either without effect or inhibitory for many species but a stimulation was obtained with Leucopaxillus amarus. Adenine is also chemically related to the cytokinins, known to be concerned with morphogenesis, but the effects of these compounds on the growth of mycorrhizal fungi does not appear to have been investigated, although, as noted above, they are produced by Rhixopogon roseolus. If the nondiffusible factor is equivalent to or concerned with a thigmotropic response, the abolition of its effect by adenine may therefore represent an interference with a morphogenetic response and so suggests a method of investigating why the fungus grows as a weft of pseudoparenchyma.

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C. DEFENCEREACTIONS OF HOSTTISSUE I n most physiological investigations of mycorrhizas, those on the endotrophic kind have lagged behind those on ectotrophic infections. One aspect where the reverse is true concerns chemicals affecting the spread of fungus within the host. These have been intensively studied for orchid mycorrhizas by Gaumann and coworkers (see Nuesch, 1963). Fungal infection induces the production within the host of substances which limit fungal growth. I n the case of orchids, these have been identified as the dihydrophenanthrene derivatives, orchinol and hircinol. They represent a class of compounds which plant pathologists have termed phytoalexins (see review by Cruikshank, 1965). The production of these compounds by the hosts of ectotrophic mycorrhizas has not been intensively studied but Foster and coworkers (Foster and Marks, 1967; Hillis et al., 1968) consider that tannins in the outer layer of the cortex cause hyphal distortion in mycorrhizal fungi of Pinus radiata and Pseudotsuga menziesii. I n the latter, they claim evidence t h a t the toxicity was sufficient to prevent movement of carbohydrate from the Hartig Net to the sheath or, in other cases, to prevent the formation of the Hartig Net from the sheath. Included in acetone extracts of both species were stilbenelike compounds, some of which have fungitoxic activity. The search for factors affecting fungal growth has been largely unilateral, i.e. only the effects of host metabolites on fungal behaviour have been examined. It is well known that the development of the root nodules of legumes, another symbiotic system of well defined morphological and anatomical structure, requires the interaction of both host and microbial factors (Nutman, 1965). A better understanding of such bilateral interaction in the development of mycorrhizas is required. It should be realized that none of the extracts, exudates or other factors so far found to affect mycorrhizal fungi is specific to them. They may have growth-promoting or inhibiting effects on others. A combination of factors emanating from the host, such as simple carbohydrates, vitamins, amino acids and Mfactors together with inhibitory substances, tends to select the mycorrhizal fungi which themselves produce substances that change the metabolism and morphology of the host. I n future studies of this interaction, the recently developed technique of Fortin (1966) for the synthesis of mycorrhizas on excised hypocotyl-root complexes of Pinus syhestris offers exciting possibilities for experimental work.

V. Absorption of Nutrients by Ectotrophic Mycorrhizas A. MYCORRHIZAL AND UNINFECTED ROOTSAS ABSORBING ORGANS Since many species of angiospermous and gymnospermous tree, including forest dominants, have ectotrophic mycorrhiza it has been reason-

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ably assumed that the habit has some selective advantage. This selective advantage has been believed for a very long time to lie in their mode of functioning in the absorption of nutrients from the soil. Indeed Frank himself in 1894 published the results of experiments showing that nonmycorrhizal pine and beech growing in sterilized woodland soil were smaller than those which developed mycorrhizas in unsterilized or sterilized soil. This kind of experiment, with greater or less refinement of sterilizing or inoculating procedures, has been repeated again and again over the years usually with similar results where soils of low fertility or unbalanced nutrient content were used (for reviews see Harley, 1959 or 1968b). The increased growth rate was found in many cases to be associated with an increased absorption of nitrogen-containing compounds, potassium and particularly phosphorus-containing compounds into the mycorrhizal seedlings. Hatch (1937)pointed out that the absorbing area of the root-surface of mycorrhizal seedlings was increased relative to the size of the plant and that this physical increase of area afforded some explanation of the results. The application of tracer techniques using 32P enabled Kramer and Wilbur (1949)and Harley and McCready (1950)to demonstrate that the mycorrhizas of pine and of beech absorb phosphate more rapidly on an area or weight basis than uninfectedroots. Later work confirmed their conclusions. For instance Bowen and Theodorou (1967) have shown that different kinds of mycorrhiza of Pinus radiata, although varying in their rates of absorption, accumulate phosphate more rapidly than uninfected roots. Others have shown that mycorrhizal root systems of seedling oak and pine accumulate phosphate faster than uninfected systems (Clode, 1956; Lobanow, 1960; Morrison, 1962). Besides the actual increase of surface of the mycorrhizal organs themselves, Hatch pointed out that the hyphae which radiate from them into the soil might also constitute further absorbing area. These hyphae are variable in frequency and extent in different kinds of mycorrhiza even on a single species of plant. Stone (1950)observed that those seedlings of Pinus radiata which had extensive mycelial connexions between fungal sheath and soil absorbed more phosphorus than those with few. A direct confirmation of the effcacy of these hyphae in absorption and translocation was demonstrated by Melin and coworkers in a series of papers (Melin and Nilsson, l950,1952,1953a, b, 1955, 1958; Melin et al., 1958) in which they described the uptake of isotopically labelled phosphate, nitrogen compounds, calcium and sodium by the emanating hyphae of pine mycorrhizas. The pine seedlings were grown in aseptic two-membered cultures withmycorrhizalfungito thehyphae of whichalone theisotopiccompound wasapplied. Isotopeswerefoundinallpartsof thehostincludingtheleaves. Since mycorrhizas, although they are dual organs, function as integrated absorbing systems, the same methods can be applied to their study

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as to that of roots. Experiments with excised mycorrhizas under controlled conditions have provided much information on the effects of diverse factors upon their physiological processes. Continued accumulation of ions by them is metabolically dependent as with roots. It is sensitive to oxygen, temperature, supply of organic substrates, hydrogen-ion concentration, metabolic inhibitors and other factors as in accumulation by many living plant materials. There is very little in this aspect of their study, which has been reviewed by Harley (1959, 1968b), that requires special consideration or comment here. The view that a greater respiratory rate of mycorrhizas might explain their greater ability to absorb some ions cannot be upheld as, for instance, the work of Kramer and Hodgson (1954) showed. There is no reason to ascribe to them a respiratory metabolism or electron-transport system (Harley and ap Rees, 1959) of anything but a usual kind. They appear to operate a normal Krebs cycle (Carrodus and Harley, 1968) and a dark fixation of carbon dioxide which is increased during assimilation of inorganic nitrogen or during excessive cation uptake (Harley, 1964; Carrodus, 1967). I n cation absorption, mycorrhizas select potassium preferentially to rubidium and sodium ; potassium seems to compete more directly with rubidium than sodium (Harley and Wilson, 1959, 1963). They readily absorb nitrogen as ammonia or in organic form, but have a restricted or no ability to absorb and reduce nitrate. The absorption of ammonia, which is metabolically dependent, is associated with its incorporation into organic forms of which glutamine is of prime importance (Carrodus, 1966, 1967). The striking feature of difference between mycorrhizas and roots lies in the primary site of accumulation of ions which in the former is into the fungal sheath. This aspect has been most fully examined using mycorrhizas of Pagus sylvatica which, on account of their size, are easily dissected into two parts; fungal sheath tissue and host core. The latter is comprised mainly of host tissues with a Hartig Net of fungus between its outer cells. During absorption over periods of a few hours to a day from concentrations of H232P04-below mM inconditions of adequate aeration. SO-SO% of the absorbed phosphate is found in the sheaths (Harley and McCready, 1952). Wilson (1957) gives about 60% of rubidium absorbed from 0.078-mM 86RbC1as present in the sheath immediately following a few hours’ absorption, and Carrodus (1967) reports that the products of the metabolism of absorbed ammonia are similarly retainedin the fungus. The high rate of uptake of ions from dilute solutions is therefore a reflexion of accumulation particularly in the fungal layer. Questions therefore arise about the mechanisms of the movement of ions through the fungal sheath, of therelease of the material accumulated in the sheath and its movement to the host. These questions have only been examined with respect to phosphate.

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B. MOVEMENT OF PHOSPHATE THROUGH THE FUNGAL SHEATH The two possible routes by which phosphate may pass through the fungal sheath are between the fungal cells in the interhyphal spaces and within the living hyphae. Harley et al. (1958) showed that, although phosphate passed through the interhyphal spaces when mycorrhizas were immersed in solutions of high concentration (32 mM), movement by this route was negligibly small in solutions of low concentration (0.0320.3 mM), that is in concentrations ecologically possible in soil solutions. Their method consisted of the examination of the effects of inhibitors and low temperatures on the rates of penetration of phosphate to the cores when mycorrhizas were aerated in a range of moderate phosphate concentrations (0.032-3.2 mM). I n the lowest and the highest concentrations inhibiting factors which, affect both sheath and core when free, diminished the uptake by both. I n intermediate concentrations (around 3 mM), similar inhibiting factors diminished, in the expected way, uptake by the sheath, but left unaffected or even stimulated movement to the core. The simplest conclusion was that, in these intermediate concentrations, inhibition of uptake by the sheath allowed increased diffusive movement through the interhyphal spaces so that there was an effective available concentration of free phosphate a t the surface of the host. The absence of such a n effect a t low concentrations was explicable only if diffusive movement through the sheath to the core was negligibly small during the time of the experiments (up to 2.5 hr.). The same group (Harley et al., 1954) estimated the magnitude of the pool of phosphate in the sheath with which the phosphate passing through it must mix on the way to the core. The departure from linearity of the curve of increase of radioactivity in the core in short time periods, when mycorrhizas were kept in 0.016 mM phosphate, was very small. Hence they concluded that only a small part of the phosphate in the sheath was in the route of passage to the host. Subsequent experiments by Harley and Loughman ( 1963) showed that inorganic orthophosphate passed to the host from the fungal sheath during absorption and was thereincorporated into organic phosphate compounds or stored as orthophosphate. These findings were confirmed and extended by Jennings (1964a, b) who showed not only the existence of a small pool of orthophosphate in the route of passage through the sheath but also that it might vary in magnitude with external conditions. The fate of the phosphate accumulated in the fungal sheath was examined by Harley and Brierley (1954, 1955). Mycorrhizas which had been allowed to accumulate labelled phosphat,e in their sheaths released it to the host if kept in unlabelled phosphate of lower concentration or in phosphate-free buffer. This redistributive movement was found to be 3*

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temperature- and oxygen-sensitive and hence to be dependent on aerobic metabolism of the tissues of the fungus or host or both. Brierley (1955) was of the view that, since the oxygen sensitivity of the process of movement from the fungus to the host was similar to that of uptake by the fungus, the redistribution was closely linked with fungal metabolism in particular. Criticisms have been levelled at the conclusions drawn from these experiments by Melin and Nilsson (1958) and by Lobanow (1960) because of the use of excised mycorrhizas. Melin and Nilsson (1958) showed that decapitated mycorrhizal seedlings of Pinus accumulated a greater proportion of absorbed phosphate in the sheath of their mycorrhizas than did intact seedlings. From this they argued that the degree to which phosphate was accumulated in the sheaths of excised mycorrhizas was an artefact arising out of the elimination of the transpiration stream. Indeed many have been reluctant to accept results which seem at first sight to indicate that the fungal sheath diminishes the supply of ions to the host. I n this context it is important to realize that the experiments of many workers show higher levels of accumulation of phosphate into the root systems of mycorrhizal than non-mycorrhizal seedlings (Clode, 1956; Lobanow, 1960; Morrison, 1962). Clode (1956) showed in Pinus radiata that the accumulation was primarily in the sheaths of the mycorrhizas and that the proportion of phosphate passed to the shoot was smaller in mycorrhizal seedlings. Morrison’s (1962) work was especially interesting because he placed his seedlingsin phosphate-free medium after a period of uptake. Phosphate was translocated to the shoots of both mycorrhizal and non-mycorrhizal plants ;in the former it continued for many days at an approximately steady rate but it fell away in nonmycorrhizal plants.

C. ACCUMULATION OF NUTRIENTS The model of the functioning of ectotrophic mycorrhizas which is suggested by these experiments is that the sheath tissue together with its outgoing hyphae comprises an efficient organ for phosphate absorption, and into it the phosphate is primarily accumulated although a steady supply of phosphate to the host is maintained by a route through the living hyphae. This route by-passes the storage compartments of phosphate in the sheath. I n conditions of low phosphate supply, the storage pool is mobilized and redistributed by a mechanism linked with aerobic metabolism. Such a mechanism would have selective value in soils intensely colonized by roots and micro-organisms which are subject to seasonal periods of plenty and deficiency. This model is only quantitatively rather than qualitatively different from one which might be applicable to normal roots. There are also two

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aspects in the absorption of nutrients by them-accumulation and storage in the cortex of the root and onward transmission to the shoot. Loughman (1966) is currently examining factors which differentially affect these two processes. I n barley plants as in other organisms the extent of accumulation in the root as compared with transport to the shoot may greatly vary in different circumstances. It may be 25% or lower and it may be 80% or more even in sterile conditions (Barber and Loughman, 1967). I n any event, accumulation in the root cortex is a significant part of uptake and the phosphate moving to the shoot does not pass through the storage pools (Crossett and Loughman, 1966). Mycorrhizas differ essentially from the root by possessing an extra fungal cortex into which very rapid accumulation is possible. Once trapped in the fungal tissue of the organ the nutrient is available for later utilization. Recent work which compares the growth and nutrient absorption of sterile crop plants and those with their roots normally associated with micro-organisms often show that there are differences. The results are somewhat contradictory (Subba-Rao et al., 1961 ;Barber andLoughman, 1967; Bowen and Rovira, 1966; Rovira and Bowen, 1966). Some show diminished total uptake of phosphate by unsterile roots and some an increased uptake. Others show a decreased percentage transIocation to the shoot at low external concentrations but not a t high; others show an increased movement to the tops. This kind of variability is exactly to be expected in casual infections of variably specific nature and variable intensity. It is not more than an extension of what is known of the effect on rhizosphere organisms. This does not detract from the value of such experimentation in the criticism of certain work on absorption. But, in ectotrophic mycorrhizas, not only is the fungus dominant in the rhizosphere but it has intimate and constant structural and physiological relations with its host root. Of greater interest for comparative purposes are the lichens. These closely-knit dual organisms of fungus and alga not only show great similarities to ectotrophic mycorrhizas in their carbohydrate metabolism but also in their ability to accumulate ions. They are often organisms of seasonally variable habitats, from cold polar to very hot dry deserts where they may only have few short periods suitable for active growth and metabolism each year. The work of Smith (1962) and others has emphasized their excessive ability to absorb and accumulate inorganic and organic substances rather than to assimilate and utilize them. The mechanisms by which such diverse symbiotic fungi exert such efficient absorptive and accumulating processes remains to be seen ; as does that by which ions move from the fungi to their autotrophic hosts.

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