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

Transcription

1 New Phytol. (1992), 122, A study of ageing of spruce [Picea sitchensis (Bong.) Carr.] ectomycorrhizas. II. Carbohydrate allocation in ageing Picea sitchensis/ Tylospora fibrillosa (Burt.) Donk ectomycorrhizas BY J. W. G. CAIRNEY^ AND I. J. ALEXANDERS Department of Plant and Soil Science, University of Aberdeen, Aberdeen AB9 2UE, Scotland, UK {Received 22 July 1991; accepted 10 April 1992) SUMMARY Translocation of '*C-labelled photosynthate was studied in seedlings of Picea sitchensis (Bong.) Carr. ectomycorrbizal with Tylospora fibrillosa (Burt.) Donk grown in Perspex observation chambers. Although translocation to young and older mycorrhizas was observed, accumulation of current '*C-labelled photosynthate was reduced in older compared with young mycorrhizas in the same root system. Non-structural carbohydrate components of P. sitchensis/t. fibrillosa and undefined beech ectomycorrhizas collected from the field were quantified by gas-liquid chromatography. Relative differences in accumulated carbohydrates were identified between P. sitchensis/t. fibrillosa and beech mycorrhizas. Levels of total soluble and insoluble carbohydrate were lower in older compared with young P. sitchensis/t. fibrillosa mycorrhizas. The soluble sugars arabitol, glucose and mannitol were detected in significantly lower quantities in older mycorrhizas whilst fructose and trehalose content was similar to that of young mycorrhizas. These results are discussed in relation to the physiology of the ageing mycorrhiza. Keywords: Ectomycorrhizas, ageing, carbon accumulation, translocation. INTRODUCTION Using mycorrhizas synthesized between Eucalyptus pilularis Sm. and Pisolithus tinctorius (Pers.) Ectomycorrhizal infection generally results m an Coker & Couch in growth pouches, however, increased accumulation of newly-fixed photo- Cairney ef a/. (1989) demonstrated that the ability of synthetic products in the root system as a whole, ectomycorrhizas to attract photosynthate was compared with root systems of uninfected plants of greatest soon after their formation and that there was the same species (e.g. Shtroya et al, 1962; Nelson, a progressive reduction in the amount of photo- 1964). This effect has not, however, been observed synthate translocated to them as they age. More than in all cases (Ahrens & Reid, 1973; Nylund & ninety days after inoculation of root systems with the Wallander, 1989). Within the ectomycorrhizal root fungus, no translocation of current photosynthate to system ectomycorrhizas have been shown to act as mycorrhizas could be detected by autoradiography, greater sinks for photosynthate accumulation than suggesting that fungus must then rely on stored unmfected short lateral roots (Bevege, Bowen & carbohydrate for continued physiological activity. Skinner 1975 Cairney, Ashford & Allaway, 1989), Al Abras et al (1988) described an age sequence of with greatest accumulation associated specifically mycorrhizas, presumed to be formed by a He6e/oma with the region of the mycorrhizal root where the sp., on nursery seedlings of Picea excelsa (Lam.) fungal sheath had formed (Cairney et al, 1989). Link. One- and two-year-old ectomycorrhizas lacked sheaths and external hyphae. They remained metabolically active but had reduced ability to respire ' Present address: Department of Soil Science, Waite Agricultural Research Institute, Glen O ~ d' ^A 50M. Australia. (^g^bon supplied in an external solution. ^^^^ ^^^^^^ ^^ ^^ To whom correspondence should be addressea. '

2 154 J. W. G. Cairney and I. J. Alexander defined ectomycorrhizas formed between Picea sitchensis (Bong.) Carr. and either Paxillus involutus (Batsch) Fr. or Tylospora fibrillosa (Burt.) Donk, Downes, Alexander & Cairney (1992) observed no such disappearance of the fungal sheath. Rather, ageing was associated with a reduction in extramatrical hyphae and a loss of turgidity, proceeding distaliy along the mycorrhiza. The progressive loss of turgidity was related at the ultrastructural level to degeneration of host cortical cells and subsequent degeneration of the Hartig net, root meristem and finally vascular tissue, and was accompanied in all tissues by a decline in metabolic activity measured as the potential of fresh hand-cut sections to hydrolyse fiuorescein diacetate. Downes et al. (1992) showed that although the morphological appearance of an individual mycorrhiza was not a reliable indicator of its chronological age, it was possible to recognize morphologically ' younger' (light and turgid over most of their length) and 'older' (dark and wrinkled with a stnall light apex) individuals. These morphological changes were associated with the decline in extent of the functional interface between the symbionts outlined above. In this paper we use the morphological age criteria of Downes et al. (1992) to test the hypothesis that the carbohydrate status of field-collected, young and old P. sitchensis/t. boiling 80 % ethanol for 15 min in test tubes stoppered with glass marbles. Pooled supernatants were reduced to 2 ml on a rotary evaporator at 38 C and the residue stored at 20 C until required. The reduced supernatant was deproteinized by adding 2 ml (20%) A1(OH)3 with shaking for 15 min and centrifuged at 3300 rpm for 7 min. The residue was resuspended twice in 1 ml distilled HgO, centrifuged and the pooled supernatants were de-ionized by stirring with c. 1 ml mixed resin (equal weights of AmberliteIR-120 and IRA-93,BDH Chemicals) for 15 min. Following removal of the extract, resin was washed twice with 2 ml distilled HjO and the total extract was then reduced to dryness on a rotary evaporator at 38 C. Dried samples were stored for 24 h in a vacuum desiccator prior to use. Extraction of insoluble carbohydrates. Residues from ethanol extracts were macerated in 1 ml 0-01 M citrate buffer (ph 4-5) and incubated with a further 1 ml buffer and 2 mg amyloglucosidase (AMG) (Sigma) at 55 C for 4 h with intermittent shaking. The mixture was subsequently centrifuged at 3300 rpm for 15 min, and the residue washed twice with 1 ml distilled H,O and re-centrifuged. Pooled supernatants were de-proteinized, de-ionized, dried and stored as described above. fibrillosa mycorrhizas is different quantitatively and qualitatively. Data on young beech {Eagus sylvatica L.) mycorrhizas are given for comparison. We also report on differential partitioning of current assimilate between young and Analysis of carbohydrates by gas-liquid chromato- graphy. Trimethylsilyl derivatives were prepared by the sequential addition of 70 ^1 anhydrous pyridine, 20//I hexamethyldisilazane and 10//I trichloroold P. sitchertsis/ T. fibrillosa mycorrhizas within the methylsilane (Sweeley et al, 1963). Samples were same seedling root system. shaken and stored in a vacuum desiccator at 10 C overnight. Derivatives (1 //I) were separated on a Packard 439 gas chromatograph fitted with a column MATERIALS AND METHODS packed with 2% CP SIL 8 on Chromasorb G Carbohydrate content of mycorrhizas of different age ( mesh), using a temperature programme of Collection of mycorrhizas. Roots were collected from C increasing at 6 C min"^ (initial period the surface organic horizons of a 44-yr-old stand of 3 min, final period 5 min). Picea sitchensis (Bong.) Carr. in Durris forest, N.E. Carbohydrates were identified by co-chromatoscotland (NJ ) during September-November graphy with known standards and quantified using a Mycorrhizas of Tylospora fibrillosa (Burt). Spectra-Physics 4290 integrator and comparison Donk were identified (Taylor & Alexander, 1991), with standard curves constructed for each carboexcised and stored over ice until use. Excised hydrate. Erythritol was included as an internal mycorrhizas were sorted into young (fully turgid) standard in all samples prior to the de-proteinizing and older (darkened and wrinkled to some extent, step. The values presented below have thus been but with turgid apices) individuals based on the corrected according to the efficiency of erythritol morphological descriptions of Downes et al. (1992). recoverv. Mycorrhizas totalling mg fresh weight (young) and mg (older) were obtained on each Accumulation of photosynthates in mycorrhizas of of three separate sampling occasions. different age For comparative purposes, two samples of undefined beech mycorrhizas (63 and 60 mg fresh Synthesis of mycorrhizas. Seedlings of P. sitchensis weight) were collected from the surface horizons of a were inoculated with T. fibrillosa (isolate F3 from 150-yr-old stand oi Eagus sylvatica L. at Burnhervie, mycorrhizas collected from the site described above; N.E. Scotland (NJ ) in December Taylor & Alexander, 1991), in Seed-Pack growth Extraction of soluble carbohydrates. Soluble carbo- pouches (Northrup King Co., Minneapolis, USA) as hydrates in each sample were extracted in 3 x 2 ml described by Piche & Fortin (1982) and maintained

3 Ageing of spruce ectomycorrhizas. II I5S Table 1. Mean gross carbohydrate contents of young and older Picea sitchensis/tylospora fibrillosa and Fagus sylvatica mycorrhizas, as measured by GLC. {a), {b), {c) indicate significantly different mean values {P = 0-05) based on one-way analysis of variance and computed LSD values P. sitchensis IT. (young) fibrillosa P. sitchensis 1T. (older) fibrillosa Fagus sp. mg g-i f.wt (% total) mg g~^ f.wt (% total) mg g"^ f.wt (% total) Soluble carbohydrate AMG hydrolysable carbohydrate Total carbohydrate ll-34(a) 4-07(a) 15-42(a) (74) (26) 6-31(b) 2-15(b) 8-47(b) (75) (25) ll'52(a) 9-68(c) 20-84(c) (54) (46) Arabitol Fructose Glucose Mannitol Carbohydrate Sucrose Trehalose AMG Figure 1. Concentrations of individual carbohydrates present in young (H) and older (D) sitchensis I Tvlospor a fibrillosa and Fagus sylvatica ( )' mycorrhizas as measured by GLC. (T) indicates that only trace quantities were detected, and bars give LSD values computed following one-way analysis of variance. Table 2. Mean amounts of "C {cpm±se) accumulated in sub-samples of young and older Picea sitchensis/tylospora fibrillosa mycorrhizas from the same root system. ' Time since transfer' refers to the number of weeks since seedling was transferred to peat substrate Time since transfer (wk) Young Older Ratio young:older (+1112) 3599 ( + 602) 322 (±43) 1600 (±220) 771 (±117) 1647 (±141) 66(±12) 145 (±34) 97 (±26) 439 (±23) 2-6:1 54-5:1 2-2:1 16-0:1 1-8:1 under ambient laboratory conditions. After 6-12 wk, infected seedlings were transferred to 20 x 20 cm Perspex observation chambers (Finlay & Read, 1986) containing moist peat, and incubated in a glasshouse under ambient light with photoperiod extended to 16 h by incandescent light for wk with weekly watering. Root systems were maintained in darkness at 15 C in a cooling water bath. Labelling with ^^CO^- In preparation for labelling with '^COj shoots of individual plants were sealed into a Perspex chamber fitted with inlet and outlet ports. Shoots were exposed to a photon fiuence rate of 780 /(mol m"^ s"^ and laboratory air was pumped through the labelling chamber for 5 h. ^^COa was generated by mixing 3-7 or 7-4 MBq ("C) sodium bicarbonate with an excess of 75% lactic acid in a

4 156 J. W. G. Cairney and I. J. Alexander cuvette attached to the inlet/outlet ports of the labelling chamber and circulated by pumping for 18 h under the same lighting regime. Plants were removed from the labelling chamber and allowed to stand for a further 24 h translocation period before removal of mycorrhizas. most striking difference observed in the beech mycorrhizas was the relative increase in mannitol and decrease in glucose compared with P. sitchensis/t. fibrillosa (Fig. 1). Accumulation of photosynthates Removal of mycorrhizas and determination of " C content. Apical 2 mm segments of mycorrhizas were excised and, based on the morphological age classes of Downes et al. (1992), divided into young and older mycorrhizas. Where possible, three groups of ten mycorrhizas were collected in each age class from each plant. Groups of ten mycorrhizas were placed in scintillation vials with 10 ml Picofluor 40 scintillation fluid (Packard), and "C content measured using a Packard Tri-Carb 4000 liquid scintillation counter. Data were not corrected for the amount of "C fixed for the shoots of different plants. RESULTS Young mycorrhizas of P. sitchensis/ T. fibrillosa invariably accumulated more '*C than older mycorrhizas in the same root system (Table 2), indicating that the former act as the greater sinks for newly fixed assimilates. The absolute amounts of ^''C accumulating in young mycorrhizas was highly variable between individual plants. Accumulation in older mycorrhizas was not consistently proportional to accumulation in the young organs, but in plants labelled wk after transfer to peat substrate in observation chambers the ratio of young;old was relatively consistent (l-8-2-6;l). Plants transferred to fresh peat substrate 38 wk prior to labelling showed proportionally less accumulation in older mvcorrhizas. Carbohydrate analysis Young mycorrhizas of P. sitchensis/ T. fibrillosa contained approximately twice as much total nonstructural carbohydrate per unit fresh weight as older mycorrhizas in the same root samples (Table 1). The major soluble carbohydrates of young mycorrhizas were identified as glucose, mannitol, fructose, trehalose and, to a lesser extent, arabitol (Fig. 1). Sucrose was not detected in any of the samples. A similar range of soluble sugars was present in older mycorrhizas, although, with the exception of fructose and trehalose, the concentrations per unit fresh weight were significantly lower in older than in young mycorrhizas (Fig. 1). Concentrations of AMG-hydrolyzable carbohydrates, taken to represent starch and/or glycogen reserves, were also lower in older mycorrhizas. The relative contribution of AMG-hydrolyzable carbohydrates to the total carbohydrate {c.2s%) was, however, similar in both classes of mycorrhizas (Table 1). Whilst the absolute concentrations of individual soluble carbohydrates generally decreased in older mycorrhizas, the concentrations of fructose and trehalose relative to total carbohydrate increased by some 50"(,. Concentrations of the remaining soluble carbohydrates were decreased relative to total carbohydrate. Beech mycorrhizas had a higher total non-structural carbohydrate content per unit fresh weight than those of P. sitchensis/ T. fibrillosa (Table 1). Since the total soluble carbohydrate concentration was similar to that of young P. sitchensis/ T. fibrillosa mycorrhizas this undoubtedly reflects the higher content of starch plus glycogen in the beech mycorrhizas. Within the soluble compounds, the DISCUSSION In suggesting a progressive decrease in the translocation of photosynthate to E. pilularis/p. tinctorius ectomycorrhizas as they age, Cairney et al. (1989) were mindful that the conditions imposed on their plants in growth pouches may have affected the physiology and longevity of mycorrhizas. The results reported here, where mycorrhizas were allowed to develop in peat, and could be classified based on known morphological criteria associated with ageing (Downes et al., 1992), indicate a similar decrease in translocation to older P. sitchensis/ T. fibrillosa mycorrhizas. Although "C-labelled compounds were translocated to older mycorrhizas in all plants, the proportional decrease in translocation to old versus young mycorrhizas in plants labelled 38 wk after transfer to fresh peat substrate (compared with those labelled after wk) is consistent with a progressive decrease in translocation as mycorrhizas age. Marshall & Waring (1985) proposed that translocation of carbohydrates to non-mycorrhizal roots of conifers occurs only during root formation, and they suggested that the root subsequently relies on carbohydrate stored as starch during formation for subsequent maintenance respiration. The greater longevity of ectomycorrhizas over non-mycorrhizal roots was attributed to an ectomycorrhiza-mediated reduction in root maintenance respiration (Marshall & Perry, 1987). On the contrary, our results indicate that carbon continues to be translocated to older mycorrhizas. However, it is important to note that movement of recently transported carbon compounds can occur between ectomycorrhizas con-

5 Ageing of spruce ectomycorrhizas. II 157 relative importance of these carbohydrates differed between young P. sitchensis/t. fibrillosa mycorrhizas and those of beech in our study, with a greater concentration of mannitol and no detectable arabitol in the latter. Differences in relative accumulation of trehalose and mannitol have been observed in ectomycorrhizal fungi (both ascomycetous and basidiomycetous species) in axenic culture (Martin et al, 1988). Similar interspecific differences in the relative importance of these carbohydrates have been observed in the extramatrical mycelium of ectomycorrhizal basidiomycetes (Soderstrom, Finlay & Read, 1988), emphasizing differences in carbon partitioning between individual basidiomycete species. In ectomycorrhizal basidiomycetes, trehalose is regarded as the major soluble storage carbohydrate (Martin, Canet & Marchal, 1984; Soderstrom et al, 1988). Arabitol and mannitol, on the other hand, appear to serve a largely translocatory function (Soderstrom et al, 1988). It is therefore interesting that the concentrations of arabitol and mannitol were lower in older P. sitchensis/t. fibrillosa mycorrhizas, while the concentration of trehalose was similar to that in the young mycorrhizas. This seems likely to indicate reduced translocation to the extramatrical mycelium supported by the older mycorrhizas, probably associated with lowered physiological activity at the mycelial growing front. The reduction in the amount of extramatrical mycelium associated with older T. fibrillosa mycorrhizas (Downes et al, 1992) is consistent with this. Assuming that the carbon requirement of the mycorrhiza itself is low compared to an active growing front, the fact that older mycorrhizas still contained a significant amount of carbohydrate is further evidence of lowered physiological activity at the growing front. A decrease in the size of the sink for translocated carbon at the growing front, and consequent decrease in translocation, might then result in reduced The low amounts of sucrose may also be partly synthesis of translocatory compounds in the mycorexplained by the time of year during which mycorrhiza. rhizas were sampled. E. sylvatica would not have Reduced physiological activity in the extramatrical been actively photosynthesizing during December, mvcelium and the concomitant reduction in the and photosynthesis in P. sitchensis may have been absorption of inorganic nutrients would undoubtedly low in the period September-November; this may also result in a reduction in the levels of nutrients therefore have been refiected in low rates of sucrose available for transfer to the host at the interface translocation to mycorrhizas. (although this may, for a time, be offset by utilization The hexose sugars resulting from sucrose hyof phosphate, for example, stored as polyphosphate drolysis (fructose and glucose) may be present in the in the sheath and Hartig net; Chilvers & Harley host cells, the apoplast of the interface between the (1980)). Thus older mycorrhizas, to which there is symbionts or in fungal tissue, depending largely on limited translocation of photosynthate and which the location of the mvertase(s) catalyzing the hycontain lowered levels of soluble and insoluble drolysis. Given the presumed rapid incorporation of carbohydrates, are likely to be associated with limited hexoses into fungal metabolites (Bevege et al, 1975) extramatrical mycelium activity and lowered availit is unlikely that they would be present in significant ability of transferable nutrients in the fungus. The quantity in the fungal tissue. availability of transferable nutrients at the interface Arabitol, mannitol and trehalose are regarded as may therefore be important for continued allocation characteristic fungal carbohydrates of ectomycor- of carbon to the mycorrhiza. rhizas (Martin, Ramstedt & Soderhall, 1987). The nected by a common mycelium (Finlay & Read, 1986). It might therefore, be argued that the " C accumulation observed in older mycorrhizas arose not from direct translocation within the plant, but as secondary translocation through interconnecting mycelium from young mycorrhizas. Secondary translocation to older mycorrhizas would seem unlikely, though, since connected developing mycorrhizas and actively growing extramatrical mycelium would represent much greater sinks for available carbohydrate. Concentrations of total soluble carbohydrate detected in both P. sitchensis/ T. fibrillosa and beech mycorrhizas collected from the field were similar to those previously recorded for beech mycorrhizas (Lewis & Harley, 1965). Analysis of carbohydrates in mycorrhizas from seedlings grown under similar conditions to those used for ^^CO^ labelling experiments (data not shown) indicated that concentrations were similar in these plants. Lewis & Harley (1965) found that 3 5 % of the soluble carbohydrate present in beech mycorrhizas was in the form of sucrose. Our observations that sucrose was not present in significant quantity in P. sitehensis/ T. fibrillosa mycorrhizas and only in trace quantities in beech mycorrhizas are in contrast with this. Bevege et al (1975) found that only 6-5% of carbon newly translocated to mycorrhizas of Pinus radiata was present as sucrose, suggesting that rapid transfer of sucrose to the fungus and its subsequent conversion to fungal metabolites result in low sucrose concentrations in mycorrhizas. Our ^^COg labelling experiments indicated that a significant quantity of photosynthate was translocated to young P. sitchensis/t. fibrillosa mycorrhizas, presumably as sucrose. Thereafter rapid hydrolysis of the disaccharide in the mycorrhiza to the constituent hexoses (Harley & Jennings, 1958) must be presumed to have occurred.

6 158. W. G. Cairney and I.jf. Alexander ACKNOWLEDGEMENTS This study was supported by NERC. We thank Dr B. T. Watson for permitting us to use his '*CO2 labelling apparatus. REFERENCES AHRENS, J. R. & REID, C. P. P. (1973). Distribution of 14Clabelled metabolites in mycorrhizal and non-mycorrhizal lodgepole pine seedlings. Canadian Journal of Botany 51, AL ABRAS, K., BILGER, I., MARTIN, F., LE TACON, F. & LAPEYRIE, F. (1988). Morphological and physiological changes in ectomycorrhizas of spruce (Picea excelsa (ham.) Link) associated with ageing. New Phytologist 110, BEVEGE, D. I., BOWEN, G. D. & SKINNER, M. F. (1975). Comparative carbohydrate physiology of ecto- and endomycorrhizas. In: Endomycorhizas (Ed. by F. E. Sanders, B. Mosse & P. B. Tinker), pp Academic Press, New York, London. CAIRNEY, J. W. G., ASHFORD, A. E. & ALLAWAY, W. G. (1989). Distribution of photosynthetically fixed carbon within root systems of Eucalyptus pilularis ectomycorrhizal with Pisolithus tinctorius. New Phytologist 112, CHILVERS, G. A. & HARLEY, J. L. (1980). Visualisation of phosphate accumulation in beech mycorrhizas. New Phytologist 84, DowNBS, G., ALEXANDER, I. J. & CAIRNEY, J. W. G. (1992). A study of ageing of spruce [Picea sitchensis (Bong.) Carr.] ectomycorrhizas. I. Morphological and cellular changes in mycorrhizas formed by Tylospora fibrillosa (Burt.) Donk and Paxillus involutus (Batsch. ex fr.) Fr. New Phytologist 122, FiNLAY, R. D. & READ, D. J. (1986). The structure and function of the vegetative mycelium of ectomycorrhizal plants. I. Translocation of '''C-labelled carbon between plants connected by a common mycelium. New Phytologist 103, HARLEY, J. L. & JENNINGS, D. H. (1958). The effect of sugars on the respiratory responses of beech mycorrhizas to salts. Proceedings of the Royal Society of London B 148, LEWIS, D. H. & HARLEY, J. L. (1965). Carbohydrate physiology of mycorrhizal roots of beech. I. Identity of endogenous sugars and utilization of exogenous sugars. New Phytologist 64, MARSHALL, J. D. & PERRY, D. A. (1987). Basal and maintenance respiration of mycorrhizal and non-mycorrhizal root systems of conifers. Canadian Journal of Forest Research 17, MARSHALL, J. D. & WARING, R. H. (1985). Predicting fine root production and turnover by monitoring root starch and soil temperature. Canadian Journal of Forest Research IS, MARTIN, F., CANET, D. & MARCHAL, J.-P. (1984). In vitro natural abundance *'C NMR studies of the carbohydrate storage in ectomycorrhizal fungi. Physiologie vegetale 22, MARTIN, F., RAMSTEDT, M. & SODERHALL, K. (1987). Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas. Biochimie 69, MARTIN, F., RAMSTEDT, M., SODERHALL, K. & CANET, D. (1988). Carbohydrate and amino acid metabolism in the ectomycorrhizal ascomycete Sphaerosporella brunnea during glucose utilization. Plant Physiology 86, NELSON, C. D. (1964). The production and translocation of photosynthate-'^c in conifers. In: The Formation of Wood in Forest Trees (Ed. by M. H. Zimmerman), pp Academic Press, London. NYLUND, J.-E. & WALLANDER, H. (1989). Effects of ectomycorrhiza on host growth and carbon balance in a semihydroponic cultivation system. New Phytologist 112, PICHE, Y. & FORTIN, J. A. (1982). Development of mycorrhizae, extramatrical mycelium and sclerotia on Pinus strobus seedlings. New Phytologist 91, SHIROYA, T., LISTER, G. R., SLANKIS, V., KROTKOV, G. & NELSON, C. D. (1962). Translocation of the products of photosynthesis to roots of pine seedlings. Canadian Journal of Botany Mi, SoDERSTROM, B., FiNLAY, R. D. & READ, D. J. (1988). The structure and function of the vegetative mycelium of ectomycorrhizal plants. IV. Qualitative analysis of carbohydrate contents of mycelium interconnecting host plants. New Phytologist 109, SwEELEY, C. C, BENTLEY, R., MAKITA, M. & WELLS, W. W. (1963). Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. Journal of the American Chemical Society 85, TAYLOR, A. F. S. & ALEXANDER, I. J. (1991). Ectomycorrhizal synthesis with Tylospora fibrillosa, a member of the Corticiaceae. Mycological Research 95,

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