The structure and function of the vegetative mycehum of ectomycorrhizal plants

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1 Nezv Phytol. (1988), 19, 13 1 The structure and function of the vegetative mycehum of ectomycorrhizal plants IV. Qualitative analysis of carbohydrate contents of mycelium interconnecting host plants. BY B. SODERSTROM, R. D. F INLAY AND D. J. READ Department of Botany, University of Sheffield, Sheffield SIO 2TN, UK (Received 9 October 1987; accepted 21 January 1988) SUMMARY Plants of Pimis spp. were grown in observation chambers with the mycorrhizal fungi Suillus hovinus, Pisolithus tinctorius or Paxillus itivohitus..\hev interconnecting mycelial systems had developed between plants, individual hosts in some chambers of each species were fed with "CO.^. Mycelia from radioactively labelled and unlabelk-d chambers were harvested and their carbohydrates were extracted, separated chromatographically and identified. The major carbobydrates in all of tbe fungi were trehalose, mannitol and arabitol, tbeir relative proportions differing in the different fungi. The results are discussed in relation both to carbon nutrition of tbe fungus and to carbon transfer between interconnected plants. Key words: Kctomycorrbiza, carbon transfer, carbohydrate, translocation, Pituis spp. T NTRODUf-TION Finlay and Read (198) showed that when shoots of single pine plants growing in mycorrhizal association with other pines were exposed to "CO.,, labelled assimilate moved readily through the interconnecting mycelium from 'donor' to 'receiver' plant as well as to the advancing edge of the mycelial network. Subsec^uently it was confirmed by means of micro-autoradiography that the hyphae within the mycelial strands which interconnect the mycorrhizal plants provide the conduits through which label is transferred between plants (Duddridge et al., 1988). Labelled assimilate was traced through the sheath of the receiver into host cortical tissue. However, we still have little knowledge of the identity of the carbon compounds being translocated in mycorrhizal mycelia. The pioneering studies of Lewis & Harley (195a, b) using e.xcised beech mycorrhizas showed that assimilates, mostly in the form of sucrose, move from the autotroph to the lungal tissue of the sheath where they are rapidly converted to metabolie intermediates amongst which trehalose and mannitol predominate. To date, the extent to which such compounds are subsequently involved in the processes of carbon transfer through intact mycelial systems has not been examined though Soderstrom & Read (1987) demonstrated that ectomycorrhizal mycelia were heavily dependent upon a continuous supply of current assimilate for the maintenance of respiratory activity. It is evident that, in entire mycorrhizal systems, the flow of current assimilate into the external mycelial network is likely to be of importance not only for the support of interconnected mycorrhizal plants but also as a potential source of energy-rich substrate for the entire microbial community of the soil. For this reason, it is necessary to establish the identity of the major compounds being translocated through the mycorrhizal mycelia. In the present study, the major carbohydrates occurring in the intact mycelial phase of three ectomycorrhizal fungi have been extracted and identified. Analyses were carried out both of radioactively labelled carbohydrates following exposure of 'donor' plants to "CO.^, and of unlabelled mycelium. In all cases, the fungi were freshly collected from vigorous mycorrhizal systems in which they were forming anastomosing mycelial networks between infected host plants.

2 14 B. Soderstrom, R. D. Finlay and D.J. Read MATERIALS AND METHODS Synthesis of ectomycorrhizas Rctomycorrhizas were synthesized aseptically using the methods described by Finlay & Read (198). The fungal species used were Suillus bovinus (L. ex Fr.) (). Kuntze, Pisolithus tinctorius (Mich, ex Pers.) Coker & Couch, Paxilliis involutus (Fr.) Fr., and the host plants, used in various combinations, were Pinus sylvestris L., Pinus contorta L., Pinus radiata L. and Retula pendula Roth. When mycorrhizal infection was established, the seedlings were transferred to Ferspex growth chambers (Brownlee et al., 1983; Finlay & Read, 198) with peat as substrate in the case of 5. boviyius and Paxillus involutus and colliery spoil in the case of Pisolithus tinctorius. After the mycorrhizal mycelia had colonized the whole of each chamber, uninfected seedlings were planted in the chambers and these rapidly became infected by the fungal mycelium in the solid substrate. The ectomycorrhizal mycelium thus formed an interconnecting network between the seedlings of the kind described by Read (1984) and Finlay & Read (198). The combinations of host plants and fungi used in different analyses are described below. 5. hovinus. For radiochromatographic (PC) analyses, the '""COj donors were seedlings of Pinus syhiestris interconnected with Pinus radiata. Four replicate chambers were analysed. For gas liquid cbromatographic (GLC) analysis, two replicate chambers containing Pinus contorta were used. Root tips of Pinus sylvestris infected with this fungus were also analysed by GLC. Pisolithus linctorius. For PC analyses, "CO,, was fed to Pinus sylvestris donors interconnected with Pinus contorta. Four replicate chambers were analysed. For GLC analyses, there were three replicate chambers with B. pendula as the host species. Paxillus involutus. Pinus contorta was used in all cases. One chamber was employed for '''C and one for GLC analysis. Analysis of "C-labelled carbohydrates One of the seedlings in eacb chamber was fed with 1-5 MBq of'''c which was released from sodium carbonate (NaH'^COg; specific activity 2 GBq mmol"') by addition of lactic acid. Previous tests of these chambers indicated that the bulk of the labelled '" COg was assimilated in the first 2 days after release. The chambers were incubated in a controlled environment growth cabinet for 5-7 days under the conditions described by Finlay & Read (198), so that labelled assimilates could be distributed tbrough the mycelium. After incubation, tbe chambers were opened and the mycelia were carefully removed from the substrates using fine forceps. They were immediately extracted in hot 8% ethanol. The extracts were de-proteinized by adding suspended A1(OH)3, centrifuged and deionized with equal amounts of Amberlite IR45 and IR12 (BDH). The carbohydrates in the extracts were separated by descending one way paper chromatography (PC). The solvents used were ethyl acetate/pyridine/water (8/ 2/1) and ethyl acetate/acetic acid/water (14/3/3). The chromatograms were scanned in a radiochromatogram scanner (Packard Model 722 with Digital Integrator 724) and developed using ethanolic silver nitrate. Some of the paper chromatograms were also analysed autoradiographically. Analysis of extracts from unlabelled mycelium Fxtracts of mycelium were prepared as described above. They were dried and converted to silyl (TMS) ethers and separated on a column of 2% SF52 on Diatome 'C (85-1 mesh) in a PYE 14 FID gas chromatograph using a temperature program of C min-' to 29 C, or I + 4 C min"' to 29 C for detection of glycerol and erythritol. Samples of peat and colliery spoil from all of the '''C-labelled and unlabelled chambers were also extracted and analysed for control purposes. In all cases, very low activities and carbohydrate contents were obtained, levels being between 1/1 and 1/1 of those found in the mycelial extracts. It would be most satisfactory to express tbe results in relation to fungal biomass. Unfortunately mycelium could not be recovered sufficiently cleanly from the peat to enable gravimetric determinations to be carried out, and preliminary analyses of tbese mycelial.systems have shown that interspecific differences in N-acetylglucosamine content are too large to enable meaningful assays of this compound to be used as a basis for comparison. All values were therefore expressed as a percentage relative to that of trehalose because this compound was consistently present in large amounts in all fungi. RESULTS The most important carbohydrates identified by botb tbe PC and GLC methods were the disaccharide, trehalose, and the polyols, mannitol and arabitol (Table 1), tbe relative proportions of the compounds being different in the three fungi. In the strands and mycelium of 5. bovinus, tbe most abundant labelled sugars, in descending order of activity, were arabitol, mannitol and trehalose. In the unlabelled extracts, in contrast, trehalose was the dominant compound, mannitol and arabitol being very much reduced in importance. Since the "C labelling is likely to be primarily localized in the most recently transferred carbon, this diff'erence may indicate that mannitol and arabitol are tbe

3 Carbohydrates in ectomycorrliizal mycelium 15 Table 1. Relative concentrations of, and ''C incorporated into, ethanol-extractable carbohydrates in mycelia of three species of ectomycorrhizal fungus grown in observation chambers with pine or birch. Extracts of ^"^C-labelled tissue were analysed by paper chromatography and of non-labelled tissue by gas liquid chromatograpliy {GLC). All amounts are presented relative to those of trehalose (see text) Fungus Suillus bovinus Pisolithus tinctorius Paxillus involutus Carbohydrate '"C N = 4 GLC N = 2 ' 'C n = 4 GLC H=3 "C = 1 GLC n = 1 l'rehalose Sucrose Mannitol Glucose Arabitol Erythritol Trace Trace , Standard deviation. compounds involved in translocation in this fungus, trehalose being more important as a storage compound. Low amounts of sucrose and glucose were also tentatively identified in the mycelium of 5. bovinus but further analyses would be required to confirm their identities. Sucrose has been identified as a component of the translocation stream in mycelial strands of Serpttla lacri?nans (Brovvnlee & Jennings, 1981) and Armillaria mellea (Granlund et al., 1985). Both sucrose and glucose were, however, detected in much greater quantity in the mycorrhizal root tips where they were probably located primarily in the bost tissue. In both the labelled and unlabelled extracts of Pisolithus tinctorius, arabitol was the predominant carbohydrate, its activity and concentration being more than double that of trebalose with mannitol occurring m only small amounts. The situation in the mycelium oi Paxillus involutus was the reverse of that seen in Pisolithus tinctorius, arabitol being relatively unimportant and mannitol being the predominant sugar in both the labelled and unlabelled extracts with a concentration and activity approximately three times that of trehalose. Comparisons of the relative specific activities of the carbohydrates confirm that there is more active metabolism of the incoming carbon to polyols in S. bovinus than in tbe other two fungi. DISCUSSION Finlay & Read (198) showed that '^C-labelled assimilate was translocated at rates in excess of 2 cm h"' through tbe mycorrhizal mycelium from tbe host towards the advancing hyphal front. The present study reveals that the bulk of the translocated material is likely to be in the form of trehalose, mannitol and arabitol, the relative importance of the three compounds being different in the fungi exannined. However, since trehalose is known to be the typical storage carbohydrate of fungi, it seems likely that mannitol and arabitol will be the compounds most involved in translocation. Unfortunately it was not possible to determine the intensities of labelling of the different carbohydrates in this study. If they were all labelled uniformly, tbe molar concentration of labelled mannitol and arabitol relative to that of trehalose would be twice that given in Table 1. Tbis furtber emphasizes the difference between the data obtained by GLC and radiochromatogram analysis. It is interesting that when sclerotia of Paxillus involutus were analysed separately they were found to contain considerably higher relative amounts of trehalose than were present in the mycelium (data not shown). All of the indications are, therefore, that mannitol and arabitol have an important role as translocatory compounds. The spectrum of compounds isolated is similar to that reported in studies of isolated mycorrhizal roots, although arabitol does not appear to bave been found previously in roots. It was, however, identified in fruit bodies of 5M?7/Msouj«Mx(Frerejacque, 1943). In the study of Lewis & Harley (195 a) trehalose, mannitol and glycogen constituted the main labelled compounds accumulating in the sheath of Eagus mycorrhizal root tips supplied with "C-labelled sucrose. Bevege et al (1975) found trehalose to be the dominant ethanol-extractable carbohydrate in mycorrhizal root tips of Pinus radiata infected by Rhizopogon luteolus, where it constituted approximately 5% of all radioactive carbohydrates following feeding of the host with '''CO2. The second most common carbohydrate in their extracts was glucose (approximately 2%). However, since plant tissue was also included in the extraction it is likely that the glucose was of host origin. A similar spectrum of compounds has been reported from tbe translocating structures of woodrotting fungi. Brownlee & Jennings (1981, 1982)

4 1 B. Soderstrom, R. D. Finlay and D. jf. Read studied translocation of carbohydrates in the strands and mycelium of Serpula lacrimans and found trehalose to be the dominant carbohydrate in mycelial strands where it constituted 84",, of all detected carbohydrates, and was also very abundant in the whole mycelium. They therefore concluded that this was the major translocated carbohydrate, although arabitol was also abundant close to the mycelial margin. Granlund et al. (1985) reported that erythritol was the dominant carbohydrate in the rhizomorphs of Arniillaria mellea though it was not ascertained whether this, or one of the other typical fungal carbohydrates present in the rhizomorphs, was primarily involved in translocation. The results obtained from the three ectomycorrhizal fungal species analysed in the present study and from Rhizopogon (Bevege et al., 1975) support the hypothesis presented by Lewis & Harley (195), based upon analyses of excised beech mycorrhizas, that host carbohydrates are transferred to the fungal tissues where they are quickly converted to fungus-specific carbohydrates which accumulate in a form not readily available to the adjacent host tissue. This pattern of events establishes a gradient which permits continuous transfer of carbon from host to fungus. Reciprocal transfer of carbohydrates between host and mycobiont has also been reported by several workers (Reid & Woods, 199; Read, Francis & Finlay, 1985; Duddridge et al., 1988). The form in which carbohydrates occur in the fungal tissue will be important in determining the balance of this transfer and it is clearly of interest to determine which carbon compounds move from fungus to host. The polyol, mannitol, is frequently reported in plants as well as fungi and has, indeed, been reported to occur in green pine needles (Theander, 1982). If a concentration gradient of this substance occurs between the fungal and host tissue, slow transfer from fungus to host might be possible in a way similar to that suggested by Lewis & Harley (195 ft) but in the reverse direction. An alternative possibility is that ionised carbon compounds in particular amino acids, which were not analysed in the present study, are translocated both through the mycelium and into the host plants. Indirect evidence of glutamine transfer from the mycorrhizal sheath to host tissues has been provided from studies of Reid & Lewis (Lewis, 197). Similarly, a conceptual model of carbon and nitrogen interactions in ectomycorrhizas (France & Reid, 1983) suggests that ammonium may be rapidly incorporated into glutamic acid and glutamine, the carbon skeletons being derived from the compounds, trehalose and mannitol which are shown in the present work to be the important carbohydrates in the translocation stream. It is clearly necessary to determine the relative importance of the neutral and charged components both in the process of translocation and in that of carbon transfer from fungus to receiver plant. Such determinations are currently being carried out using '^C- and ' '^N-labelled compounds as tracers in interlinked mycelial systems. REFERENCES BEVEGI-, D. L., BOWRN, G. U. & SKINNER, M. F. (1975). Com- parative carbohydrate physiology of ccto- and endo-mycorrhizas. In; Endomycorrhizas (Ed by F. E..Sanders, B. Mosse & P. B. Tinker), pp Academic Press, London, BUOWNI-EE, C, DUDDKIDGE, J. A., MALinARI, A. & READ, D. J. (1983). The structure and function of mycelial systems of ectomycorrhizal roots with special reference to their role in lorminj^ interplant connections and providing pathways for assimilate and water transport. Plant and Soil 71, BROWNI.EE, C. & JENNINGS, D. H. (1981). The content of soluble carbohydrates and their translocation in mycelium of Serpula lacrimans. Transactions of the British Mvcniogical Society 77, 1.S 19. BROWNi.riE, C. & JENNING.S, D. H. (1982). Long distance translocation in Serpula lacrimans: velocity estimates and the continuous monitorinj^ of induced pertubations. Transactions of the British Mycological Society 79, DlIUDRIDGE, J. A., FiNI.AV, R. D., READ, D. J. & B..SOIJERSTROM (1988). The structure and function of the vegetative mycelium of ectomycorrhizal plants. III. Ultrastructural and autoradiographic analysis of inter-plant carbon distribution through intact mycelial systems. New PhytologisI, 18, FiNi.AY, R. D. & READ, D. J. (198). The structure and function of the vegetative mycelium of ectomycorrhizal plants. L Translocation of '''C-labelled carbon between plants interconnected by a common mycelium. Nezv Phvtolo^ist FRANCE, R. P. & REID, C. P. P. (1983). Interactions of nitrogen and carbon in the physiology of ectomycorrhizae. Canadian Journal of Botany 1, FREREJAI-QUE, M. (1943). Sur la presence de D-arabitol dans Boletus hovimis L. Comptes rendus Acadeviie de Seances, Paris 217, GRANI.UND, H. 1., JENNINGS, D. H. & THOMPSON, W. (1985). Translocation of solutes along rhizomorphs of Armillaria mellea. Transactions of the Britisit Mveologieal Societv, LEWIS, D. H. (197). Interchange of metabolites in biotrophic symbioses between angiosperms and fungi. In: Perspectii'es in En7'iromental Biology, vol. 2, Botany (lid. by N. Stitht'riand), pp Pergamon Press, Oxford. LEWIS, D. IL & HARI.EY, J. L. (195a). Carbohydrate physiology of mycorrhizal roots of beech. II. Utilization of exogenous sugars by uninfected and mycorrhizal roots. Netu Phytologist 4, LEWIS, D. II. & HAKI.EY, J. L. (195i). Carbohydrate physiology of mycorrhizal roots of beech. III. Movement of sugars hetween host and fungus. Nezu Phytologist, READ, D. J. (1984). The structure and function of the vegetative mycelium of mycorrhizal roots. In; The Ecology and Physiology of the Fungal Mycelium British Mycological Society Symposium, vol 8. (Ed. by U. H. Jennings & A. D. M. Rayner), Cambridge University Press. REID, C. P. P. & WOODS, F. W. (199). Translocation of '^C- labelled compounds in mycorrhiza and its implications in interpreting nutrient cycling. Ecology 5, SKINNER, M. F. & BowiiN, G. D. (1974). The uptake and translocation of phosphate by mycelial strands of pine mycorrhizas. Soil Biology and Biochemistry, S3 5. SODERSTROM, B. & READ, D. J. (1987). The respiratory activity of intact and excised ectomycorrhizal mycelial systems growing in unsterilised soil. Soil Biology atid Biochemistry 19, 231. THEANDER, (). (1982). Ilydrophobic extractives from the needles of Scots pine and Norway spruce. Srensk Papperstidning 1982, 4 8.

Department of Plant and Soil Science, University of Aberdeen, Aberdeen AB9 2UE, Scotland, UK {Received 22 July 1991; accepted 10 April 1992)

Department of Plant and Soil Science, University of Aberdeen, Aberdeen AB9 2UE, Scotland, UK {Received 22 July 1991; accepted 10 April 1992) New Phytol. (1992), 122, 153-158 A study of ageing of spruce [Picea sitchensis (Bong.) Carr.] ectomycorrhizas. II. Carbohydrate allocation in ageing Picea sitchensis/ Tylospora fibrillosa (Burt.) Donk