A RELATIONSHIP BETWEEN OXYGEN TRANSPORT AND THE FORMATION OF THE ECTOTROPHIC MYCORRHIZAL SHEATH IN CONIFER SEEDLINGS

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1 New Phytol. (1972) 71, A RELATIONSHIP BETWEEN OXYGEN TRANSPORT AND THE FORMATION OF THE ECTOTROPHIC MYCORRHIZAL SHEATH IN CONIFER SEEDLINGS BY D. J. READ AND W. ARMSTRONG Department of Botany, University of Sheffield and Department of Botany, University of Hull {Received 13 Janwarj 1971) SUMMARY The response of the mycorrhizal fungus. Boletus variegatus, to living coniferous roots and to hollow silicone rubber 'roots' in anaerobic media was investigated. IMycelial macerates were cultured in nutrient and non-nutrient agar media in tubes. The roots of whole seedlings or silicone 'roots' were placed in the media to a depth of several centiaieters below the air-agar interface. The silicone 'roots' were either provided with access to the atmosphere by means of a capillary tube exposed above the agar surface, or were sealed. In young seedlings with unsuberized roots a diffuse fungal growth occurred over the whole root surface. In older, suberized, root systems, colonization was restricted to areas of the main axis through which short laterals were appearing. It was demonstrated polarographically that the area stimulating fungal growth were the only areas of significant oxygen evolution along the root. Fungal growth occurred around the silicone 'roots' only when access to air was provided by the capillary. It took the form of a diffuse mycelial ball in non-nutrient medium but, in nutrient agar, of a tight sheath with a distinct tendency for pseudoparenchyma formation. The possible role of oxygen evolution from roots is discussed both in terms of the initial stimulus tc the symbiont and of the continued physiological activity of the mycorrhiza. INTRODUCTION While advances have been made in the elucidation of the physiological role of mycorrhizal roots, considerable doubt still surrounds the nature of the initial stimulus responsible for the formation of the ectotrophic mycorrhizal sheath. Melin (1963) has shown that the growth of mycorrhizal fungi can be stimulated by both diffusible and non-diffusible factors produced by roots, but that none of these factors can initiate the characteristic Ganges normally associated with sheath formation. Harley and Lewis (1969) suggest that the peculiar growth form of the fungus on the root surface may be a thigmotrophic ' esponse. Young roots of coniferous seedlings growing in media of depleted oxygen supply may <'\ olve sufficient oxygen to produce an aerobic environment over the root surface (Armstrong and Read, 1972). In view of the normally low oxygen status of many coniferous forest soils and of the strongly aerobic requirements of the mycorrhizal fungi, experiments were designed to investigate the possibility that oxygen evolution might provide a stimulus to such organisms in the vicinity of the roots. I'iving and simulated silicone 'roots' were used in the experiments together with the "ncorrhizal fungus Boletus variegatus. Silicone 'roots' were employed in order to d'tferentiate between a chemotrophic response such as might be produced by a seedhng ^"d a purely physical thigmotropic response. 49

2 50 D. J. READ AND W. ARMSTRONG MATERIALS AND METHODS Seedling culture. Seed of Pinus contorta and Picea sitchensis was surface sterilized by the method described by Armstrong and Read (1972) and germinated on distilled water agar. Cultures of coniferous seedlings were then established in tubes containing both nutrient and non-nutrient agar media inoculated with the mycorrhizal fungus. Boletus variegatus. The nutrient medium was a 0.5% distilled water agar containing 1.0% glucose and 0.5% yeast extract. The non-nutrient medium was 0.5% distilled water agar. The medium was freshly autoclaved in boiling tubes and allowed to cool to 40 C when I ml of a mycelial macerate of B. variegatus was added to the tube. The tube was vigorously rotated to disperse the inoculum and the seedling was added just before the agar set. One series of seedlings was transferred to the inoculated medium as soon as the cotyledons emerged from the seed, at which time the hypocotyl was approximately 3 cm. A second series was first grown for a period of six months on a complete mineral nutrient medium by which time the roots had developed a normal system of short laterals. These relatively mature seedlings were transferred to the inoculated medium at this stage. The seedlings were maintained in a growth cabinet providing an 18-hour day and a light intensity of 1500 foot candles for a period of three weeks. During this period the roots were shielded by black paper. The pattern of fungal colonization was then determined for the two types of seedling and, after extraction of the seedlings from the inoculated medium, oxygen evolution rates were determined for various parts of the roots using the methods described previously (Armstrong and Read, 1972). Simulated root cultures. In order to simulate a seedling growing in an anaerobic medium, lengths of fine silicone rubber tubing were employed. This material is of uniform matric structure and gives rates of oxygen diffusion comparable with those of some herbaceous plant roots (Armstrong, 1967). One-cm lengths of this tubing were sealed at one end with solid glass rod and were fitted at the other end with a length of glass capillary tube or glass rod sufficient to reach the atmosphere above the agar surface. These systems were sterilized and placed into the two types of inoculated media used for the seedling cultures. In each case the silicone root was situated well below the agar surface. In the case of a silicone root fitted with a glass capillary projecting into the atmosphere, an internal supply of oxygen to the 'root' was assured, whereas, when fitted with a fine rod or a sealed capillary, no such supply was possible. These cultures were maintained under the same conditions as the seedlings and were examined after 3 weeks. RESULTS Seedling cultures In the seedling cultures, extensive growth of the fungus occurred on the surface of the agar exposed to air. The pattern of root colonization by the fungus depended upon the age of the root. Young seedlings stimulate diffuse growth of the fungus over the whole length of the hypocotyl (Plate i. No. i.). There was some reduction of the stimulus in the vicinity of the root apex. The seedlings of the older series with heavily suberized main axes and abundant short roots showed a different pattern of infection. The primary root in this case did not stimulate fungal growth, but hyphal complexes developed at points along the main axis where lateral roots were breaking through the cortex. The greatest stimulus was shown

3 Oxygen and mycorrhizal sheath formation 51 where lateral roots were just beginning to protrude through the cortex of the main axis. More mature laterals produced less stimulus to fungal growth (Plate i. No. 2). Simulated root cultures The pattern of fungal development was quite distinctive in all the tubes (Plate i, Nos. 3 and 4; Plate 2, Nos. 5 and 6). There were only two areas of extensive mycelial growth; the surface of the agar and around those siiicone 'roots' in which the capillary was open to the atmosphere above the tube. In those with closed capillaries or glass rods, no growth of the fungus occurred. The presence of glucose and yeast extract in the medium had a profound effect on the form of the fungus around the 'root' (Plate 2, No. 6). The fungal development was strongly reminiscent of the type of ectotrophic mycorrhizal sheath formed on many coniferous roots (Plate 2, Nos. 7 and 8) namely a partly pseudo-paranchymatous weft of hyphae developed close to the surface of the 'root' while extending outwards into the agar was a zone of more diffuse growth. In the non-nutrient medium a diffuse ball of hyphae developed around the 'root' (Plate I, No. 4) and extended into the medium. There was no aggregation of hyphae at the 'root' surface or any other feature reminiscent of sheath formation. Oxygen evolution from roots of seedling cultures In view of the dependence of fungal growth on oxygen supplies which is revealed by the siiicone root experiments, it may be assumed that fungal growth on the seedling roots must be at least partly facilitated by oxygen diffusion from the roots. Estimates of oxygen diffusion rate from different parts of the root confirmed that oxygen evolution was occurring from the inoculated roots. Of greater interest was the observation that only those areas showing oxygen evolution supported fungal growth. The data are presented in association with Plate i. No. 2. The results show that in the youngest seedlings a slow diffusion is in progress over most of the surface, and that the process of suberization progressively restricts oxygen release until, finally, escape occurs only through breaks in the cortex. DISCUSSION Some of the factors leading to the production of pseudo-parench)'matous masses of hyphae in sclerotia and rhizomorphs have been investigated by Garrett (1953) and Townsend (1957). These hyphal aggregates appear to be initiated by a combination of thigmotropic and chemotropic responses. There has been an absence of comparable information in connection with mycorrhiza formation. It is therefore of considerable interest that the Jcsponse to siiicone 'roots' supplied with oxygen is the formation of a sheath which is Similar in structure to that found in nature on many coniferous roots. The production of a sheath on a synthetic root helps to explain some of the factors which may be responsible for sheath formation in nature. The sheath appears to be formed as a response to three factors. Firstly, there is the localized source and probable limiting supply of one factor, Jn this case oxygen. Secondly, there is the physical presence of the siiicone which may stimulate thigmotropic responses. Thirdly, the important role of subsidiary nutrients is demonstrated by the failure to form a sheath in water agar. None of these factors appear ^0 be independently capable of stimulating sheath formation. Since fungal growth occurred upon the root surfaces of seedlings growing in the same "tedium as the siiicone roots and at the same depth, it can be assumed that oxygen evolu-

4 52 D. J. READ AND W. ARMSTRONG tion is once again playing a stimulatory role. In both types of seedling, the areas of oxygen evolution are those which would normally be concerned with nutrient uptake in the natural environment. Thus a mechanism exists which could be responsible for the establishment of the mycorrhizal sheath on absorbing root surfaces. If, under natural conditions, a number of diffusible substances are responsible for the attraction of mycorrhizal associates, the relative importance of internally transported oxygen will depend on the oxygen status of the soil in which the roots are growing. Observations of Brierley (1955) suggest that in beech litter sufficient exogenous oxygen exists for the maintenance of the mycorrhizal association. Measurements of the total air porosity of coniferous humus layers by Rennie (1956) on the other hand suggest that available oxygen in this environment may be extremely scarce through much of the year. Under such circumstances the evolution of oxygen from coniferous roots might be of importance not only for the initial attraction and establishment of mycorrhizal relation but for the efficient physiological function of the organ in nutrient absorption. These processes in beech mycorrhiza are extremely sensitive to reductions of oxygen supply (Harley et al, 1956). The maintenance of a regular, internally transported supply of oxygen would facilitate uninterrupted physiological activity by the mycorrhiza independent of external oxygen sources. Since such an internal pathway has been shown to exist (Armstrong and Read, 1972), it is possible to view the physiologically active mycorrhiza as a sink towards which oxygen absorbed by the shoot would pass. REFERENCES ARMSTRONG, W. (1967). The use of polarography in the assay of oxygen diffusing from roots in anaerobic media. Physiologia PL, 30, 540. ARMSTRONG, W. & READ, D. J. (1972). Some observations on oxygen transport in conifers seedlings. Netc Phytol., 71, 55. BRIERLEY, J. K. (1955). Seasonal fluctuations of the concentrations of oxygen and carbon dioxide in the litter layer of beech woods with reference to salt uptake by excised mycorrhizal roots of the beech. J. EcoL, 43, 404. GARRETT, S. D. (1953). Rhizomorph behaviour in Armillaria mettea. I. Factors controlling rhizomorph initiation by A. mellea in pure culture. Ann. Bot., N.s. 17, 63. HARLEY, J. L., MCCREADY, C. C, BRIERLEY, J. K. & JENNINGS, D. H. (1956). The salt respiration of excised beech mycorrhizas. IL The relationship between oxygen consumption and phosphate absorption. JVw Phytol., 55, 1. HAKLEV, J. L. & LEWIS, D. H. (1969). The physiology of ectotrophic mycorrhizas. Ad. Microbiol. Physiol, 3, 53- MELIN, E. (1963). Some effects of forest tree roots on mycorrhizal basidiomycetes. In: Symbiotic Associations (Ed. by P. S. Nutman and B. Mosse). Cambridge University Press, London. RENNIE, P. J. (1956). Some physico-chemical properties of moorland soil as related to afforestation. Ph.D thesis. Oxford University. TowNSEND, B. B. (1957). Nutritional factors influencing production of sclerotia by certain fungi. Ann. Bot- N.S., ai, 153. EXPLANATION OF PLATES PLATE I No. I. Young seedling of Pinus contorta in inoculated agar showing diffuse colonization of the whole root surface by the fungus Boletus variegatus. Colonization is seen to extend below the level of the oxygenated surface layers of agar indicating oxygen evolution from the root surface. No. 2. Part of the root system of a six-month-old seedling of Pinus contorta in inoculated agar. Intensive fungal colonization is restricted to the regions of short root formation.

5 THE NEW PHYTOLOGIST, 71, 1 PLATE I i- READAND W. ARMSTRONG-OA'lGEiV AND MYCORRHIZAL SHEATH FORMATION {jacing page 52)

6 THE NEW PHYTOLOGIST, 71, i PLATE 2 D. J. RI-:AD AND W. ARMSTRONG OXFG^iV^NZ; MYCORRHIZAL SHEATH FORMATJ

7 Oxygen and mycorrhizal sheath formation 53 Polarographic analysis of oxygen evolution from this root demonstrate that these regions are those from which oxygen evolves. Results of polarographic analysis of the root at the positions labelled are expressed as oxygen diffusion current (/(A). The residual current is o.io ^A and root temperature 3 C (see Armstrong and Read (1972) for details of method). A,0.40//A (significant Oj evolution); B,o. 10//A(noOjevolution); C,o.io//A(noOjevolution); D, 0.65 ^A (significant Oj evolution); E, 0.40 fih. (significant Oj evolution). No. 3. Silicone 'root' in inoculated non-nutrient agar. This 'root' is blocked at both ends with glass rod so that the 'root' has no oxj-gen supply. No fungal colonization of the 'root' occurs. Note the fungal growth at the oxygenated air-agar interface. No. 4. Silicone 'root' in inoculated non-nutrient agar with the 'root' attached to a capillary tube exposed to the atmosphere above the agar surface. The fungus develops as a loose globular growth in the zone of Oj evolution, and at the air agar interface. PLATE 2 No. 5. Silicone 'root' in inoculated nutrient agar. In this 'root' the capillary tube extends into the atmosphere but it is sealed at the tip. No fungal growth occurs in the vicinity of the 'root'. Once again extensive growth occurs at the air-agar interface. No. 6. Silicone 'root' in inoculated nutrient agar. This 'root' is fitted to an open capillary tube allowing oxygen supply to the silicone. Note the formation of the tight mycorrhiza-like sheath around the 'root'. No. 7. Transverse section through a sheath formed on a silicone 'root' supplied with oxygen in nutrient agar. No. 8. Transverse section of a sheath formed under natural conditions on a root of Pinus contorta.

EFFECT OF VESIGULAR-ARBUSCULAR MYCORRHIZAS ON GROWTH OF GRISELLNIA LITTORALIS (CORNAGEAEj BY G, T, S, BAYLIS

EFFECT OF VESIGULAR-ARBUSCULAR MYCORRHIZAS ON GROWTH OF GRISELLNIA LITTORALIS (CORNAGEAEj BY G, T, S, BAYLIS Botanv Dept., University of Otago, Neiv Zealand {Received 25 July 1958) (With I figure in the