Mycologia, 94(2), 2002, pp. 190199.

2002 by The Mycological Society of America, Lawrence, KS 66044-8897

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C-NMR study of exudation and storage of carbohydrates and amino acids in the ectomycorrhizal edible mushroom Cantharellus cibarius
of the surrounding medium. For instance, trehalose production has been considered as a possible tolerance response when fungi grow under stress conditions (Mellor 1992). The golden chanterelle, Cantharellus cibarius Fr., is an economically important edible ectomycorrhizal mushroom (Danell 1999, Watling 1997), phylogenetically distant from the euagarics such as Agaricus, Laccaria or Boletus (Hibbett et al 1997). The genetic differences may imply also differences in physiology and ECM formation processes in comparison with other mycorrhizal basidiomycetes (Danell 1999). The comprehension of the physiology of C. cibarius is an important step towards optimized articial cultivation. The problems in obtaining pure cultures of C. cibarius due to bacterial contamination (Straatsma et al 1985, Danell 1994a) explain why such studies are scarce. Previous attempts to nd exudates that could explain the presence of large numbers of bacteria in fruit bodies have failed (Danell 1994a). Most of the physiological studies in C. cibarius have been focused on aspects of basic nutrient requirements in order to obtain axenic cultures (Straatsma and Van Griensven 1986, Danell 1994a, Danell 1999). From other studies on ECM fungi it is known that sucrose derived from photosynthesis of the host plant is degraded to glucose and fructose by the plant root invertase (Hampp et al 1995). However, the fate of glucose and fructose once assimilated by the C. cibarius mycelium is unknown, which is surprising considering the ecological and commercial importance of this mushroom (Danell 1999). We hypothesize that the chanterelle mycelia exude organic compounds, which may explain the growth of bacteria in fruit bodies. The aims of this investigation were (1) to identify the carbon compounds exuded by C. cibarius mycelia, (2) to investigate the fate of glucose, and (3) to determine variation in exudation and storage due to strain, temperature and pH. 13C-NMR is a powerful technique that has allowed improved understanding of the carbon metabolic routes of some ECM fungi. It provides the opportunity to follow the distribution of intermediates and nal products after fungal assimilation in axenic cultures (Martin 1991). This technique was therefore chosen for this study. 190

J. Ignacio Rangel-Castro1 Eric Danell

Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences (SLU), Box 7026, SE-750 07 Uppsala, Sweden

Philip E. Pfeffer
Plant-Soil Biophysics, United States Department of Agriculture-Agricultural Research Service, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA

Abstract: 13C-NMR analyses of Cantharellus cibarius growth media were performed. We found exudation of trehalose and mannitol, which may explain the phenomenon of reproducing Pseudomonas bacteria observed inside fruit bodies. Exudation varied with strain and environment. NMR analyses of stored 13C was also performed. Trehalose, mannitol, and arginine were revealed. The mannitol pathway seems to play an important role for trehalose production in this species. This is the rst study of the fate of the photosynthetically derived carbon in the highly appreciated edible ectomycorrhizal mushroom Cantharellus cibarius. Key Words: arginine, chanterelle, mannitol, metabolism, mycorrhiza, trehalose

INTRODUCTION

Studies of carbohydrate metabolism in several ectomycorrhizal (ECM) (Martin et al 1984, 1985, 1988, 1998, Hampp and Schaeffer 1995, Smith and Read 1997) and non ECM fungi (Hammond and Nichols 1976, Wannet et al 1998, 1999, 2000) have been performed in the past few years. Trehalose (alpha-D-Glucopyranosyl-(11)alpha-Glucopyranoside) and mannitol have been found as the major carbon compounds stored in mycelia of the studied ECM fungi (Martin et al 1985, 1988, 1998, Ramstedt et al 1989). Exudation of some other sugars and polyols were found by Yu-Ping et al (1999) on hyphal tips of the ectomycorrhizal fungus Suillus bovinus. Storage and exudation are two processes that might be affected by external conditions such as temperature and pH
Accepted for publication September 4, 2001. 1 Corresponding author, Email: Ignacio.Rangel@mykopat.slu.se

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Strains and culture conditions. Many previous studies based on 13C-NMR analysis were only focused on one strain and one set of incubation conditions (Martin et al 1998). To avoid results that are only applicable for one strain, and to get results of more general importance, we selected two C. cibarius strains growing 60 km apart, and used different growing conditions for one of the strains (see below). Mycelia of the strains SNGT2-A and LBCT6 were grown on solid Modied Fries Medium (MFM) (Danell 1994a) in 9-cm petri dishes for 30 d. Both strains originate from fruit bodies collected in mixed coniferous Swedish forests. Plugs of growing mycelia (5 mm diam) were placed on top of cellophane on new MFM-agar. Actively growing mycelia were transferred to 13C-glucose enriched medium devoid of fructose, after ca 7 d of incubation at 20 C (see below). Our previous 1H-NMR analyses of C. cibarius growth medium indicate that C. cibarius utilize glucose rather than fructose when these compounds are mixed (data not shown). For this study we therefore only used glucose. Labeling studies. MFM enriched with 1-13C-Glc (99 atom % 13C, Sigma-Aldrich) to a nal concentration of 23.14 mM, with a C/N ratio of 11, was prepared to study the metabolism of C. cibarius. Fructose was omitted from the medium. Three fungal plugs were transferred to each 5.5 cm-petri dish lled with 9 mL 13C-enriched-MFM. One set (three replicates) of petri dishes were incubated under standard conditions; 20 C and pH 5.5 (Straatsma and Van Griensven 1986, Danell 1994a, b). The strains SNGT2-A and LBCT6 were grown under these conditions. In another experiment, one set (three replicates) of SNGT2-A was incubated at 13 C and at pH 4.5. At the time of inoculation, only untouched pre-cultivated mycelia were used. During the growth phase the mycelia were kept without disturbance and during harvest the mycelia were removed in one move. The plates were incubated for 30 d, after which the exponential growth phase ceases (Straatsma and Van Griensven 1986). At harvest, mycelia from the same treatment were gently removed and pooled in one ask. The remaining liquid medium was lter sterilized and also pooled in one ask. Samples were frozen at 20 C, freeze dried and stored for later treatments and analyses. NMR spectroscopy. Freeze-dried samples were ground and extracted using a cold (20 C) methanol:water (70:30, v/ v) solution. The extracts were evaporated to dryness at 40 C; the residues were dissolved in D2O and carbohydrates and amino acids were examined by NMR spectroscopy. Conditions of NMR experiments were carried out as reported by Martin et al (1998). Identication of carbohydrates and amino acids were made by comparison with spectra of standards and assignments reported previously (Martin et al 1985, Martin and Canet 1986). Degree of variation of the technique is 10%.
RESULTS

halose was the main carbohydrate synthesised by C. cibarius (FIG. 1a, b, and c). The strongest resonance in the strain SNGT2-A arose from C1 of trehalose (FIG. 2a) (19% of the total 13C-NMR); C3, C5, and C6 were also labeled (FIG. 2b) and all together accounted for 29% of the whole spectrum (FIG. 1a). Mannitol was detected though it was not abundant (5%, FIG. 1a); labeled positions were C1/6, C2/5, and C3/4. Other polyols were detected as well, e.g., arabitol (5%, FIG. 1a) and erythritol (6%, FIG. 2b). Weak signals of glucose (2%) and the Krebs cycle intermediates citrate (2%) and malate (2%) were observed as well (FIG. 2b). The spectrum of LBCT6 also indicates that the strongest signal came from trehalose C1 (FIG. 3a) (20% from the whole NMR). C3, C5, and C6 were also labeled (FIG. 3b). The chemical shifts from trehalose corresponded to 35% of the labeled assimilated 13C (FIG. 1b). Labeled mannitol was detected in higher proportion than in strain SNGT2-A, i.e., C1/ 6, C2/5, and C3/4 produced signals that represented 10% of the labeled assimilated 13C (FIG. 1b). Peaks of arabitol (5%) and malate (1%) were also observed (FIG. 3b). The NMR resonance of carbohydrates from SNGT2-A incubated at different temperature and pH (13 C and pH 4.5) showed that trehalose C1 (FIG. 4a) was the most abundant labeled component (26%) and together with C2, C3, C5, and C6 accounted for 35% of the labeled assimilated 13C (FIG. 1c). C3/4 and C2/5 of mannitol (FIG. 4b) showed a weak labeling (3%, FIG. 1c). Arabitol (FIG. 1c), citrate and glucose (FIG. 4b) produced 5%, 2%, and 12% of the total NMR respectively. Amino acid biosynthesis. The amino acids inside SNGT2-A and LBCT6 accounted for 49% of the labeled assimilated 13C (FIG. 1a and b). In SNGT2-A, arginine accounted for 24% of the labeled assimilated 13C (FIG. 1a). A similar distribution of 13C in the positions C2, C3, and C4 of glutamine was observed (FIG. 2b). Glutamine accounted for 16% of the labeled assimilated 13C (FIG. 1a), while glutamate only accounted for 6% (FIG. 2b). A weak resonance of asparagine was observed (3%, FIG. 2b). The spectrum of LBCT6 in FIG. 3a indicated that arginine was the most strongly labeled amino acid (33%, FIG. 1b), almost half of it arose from the C4; C2, C3, and C5 were also labeled. Glutamine C2, C3, and C4 accounted for 14% (FIG. 1b) while asparagine represented only 2%. When SNGT2-A was cultivated at a lower temperature and pH, the proportion of amino acids accounted for 43% of the labeled assimilated 13C (FIG. 1c). In this case there were no striking differences

Carbohydrate metabolism in mycelia of Cantharellus cibarius. NMR of 13C labeled mycelia showed that tre-

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FIG. 1. Proportion of 13C stored in mycelia of two strains of Cantharellus cibarius and two different growing conditions. * Pool of total labeled amino acids. ** Grown at pH 5.5 and 20 C. *** Grown at pH 4.5 and 13 C. The results come from pooled samples, therefore variation within each treatment was not calculated. Percentages illustrate signal intensity and not concentrations of the compounds. Mmannitol; Ttrehalose; Aarabitol; argarginine; glnglutamine.

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FIG. 2. 13C-NMR spectra from mycelium of SNGT2-A grown at standard conditions. FIGURE 2a shows the full length. FIGURE 2b shows the detailed spectrum to the right from G1 in FIG. 2a. Gglucose; Ttrehalose; Mmannitol; arg arginine; glnglutamine; gluglutamate; Eerythritol; asnasparagine; Citocitrate; Malmalate; Aarabitol.

between arginine (12%, FIG. 1c), glutamine (16%, FIG. 1c), and glutamate (13%, FIG. 4b). Labeled alanine that was hardly detected under standard incubation conditions increased to 2% in this case. C-compounds exuded in the liquid medium. Apart from residual 13C-Glc, mannitol and trehalose were found as exudates (FIG. 5). Other exudates in SNGT2 were a group of polysaccharides, while LBCT6 exuded arabitol. FIGURE 6 shows equal proportions of mannitol and trehalose in SNGT2-A, while in LBCT6 mannitol is ca 4 times higher than trehalose. When SNGT2-A was incubated at lower temperature and pH, exudation of mannitol and trehalose decreased ca 75 and 90% respectively. Also the fate of 13C in mannitol was different. In SNGT2-A it was found in positions C1 and C6, while in LBCT6 it was observed in C3/C4 (FIG. 5a and b). SNGT2-A incubated at low temperature and pH exuded mannitol labeled at positions C3/C4 and C2/5 (FIG. 5c).
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DISCUSSION

The results of the present study indicate that trehalose and mannitol were exuded in different proportions based on 13C spectra labeling patterns in C. cibarius. These patterns also depended on the strain and growing conditions. Until now, there is no experimental basis for explaining how millions of bacteria can reproduce inside long-lasting fruit bodies of chanterelles without damaging hyphae (Danell et al 1993, Danell 1994a). Yu-Ping et al (1999) showed exudation of carbon compounds from Suillus bovinus, among them mannitol. These compounds could be used by the so-called mannitol-specialized bacteria associated with S. bovinus (Timonen et al 1998). Trehalose and mannitol exuded by chanterelle mycelia are possible carbon sources for soil or fruit body bacteria (Danell et al 1993). We propose that the chanterelle bacteria would use the fungal exudates for growth and reproduction along vegetative hyphae or inside fruit bodies. It is very difcult to estimate the

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FIG. 3.

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C-NMR spectra from mycelium of LBCT6. FIGURE 3a and b as in FIG. 2. Abbreviations as in FIG. 2.

concentrations exuded, and therefore to evaluate the signicance of the exudates in comparison with other sources. However, the obser vations of bacterial growth in fruit bodies without tissue damage (Danell et al 1993) implies that there are sufcient exudates to support bacterial growth. Undamaged fruit bodies from the eld were xed for TEM by Danell et al (1993). Their pictures of bacteria showed expanded chromosomes and aggregated bacteria, indicating growth. All bacteria were distributed in the matrix between undamaged hyphae. However, the authors were unable to detect any fungal exudates due to the use of unrened methods (pers comm). Our ndings therefore suggest a possible mechanism for this co-existence. The fact that, in exudates, there were no signals from other labeled compounds found in mycelia, e.g., amino acids, indicates that it is unlikely that the presence of trehalose and mannitol was due to leakage of damaged mycelia rather than exudation. Trehalose and arginine were the most important compounds in the carbon assimilation of C. cibarius. They accounted for ca 50% of the labeled assimilated 13C-NMR depending on the strain and growing conditions. Trehalose has also been found as storage in

other ECM basidiomycetes such as Piloderma croceum (Ramstedt et al 1989) and Laccaria bicolor (Martin 1991). Other ECM fungi e.g., Cenoccocum geophilum, Sphaerosporella brunnea , and Pisolithus tinctorius (Martin et al 1985, 1988, 1998) store most of the assimilated carbon in the form of mannitol. The difference in the proportions of trehalose and mannitol stored in the two chanterelle strains could be due to a different individual storing capacity of each strain; in fact, the growth of LBCT6 is faster than SNGT2-A in similar culture conditions (Rangel et al 2000). The difference in the proportions of 13C-compounds between mycelia of SNGT2-A grown in different culture conditions (FIG. 1a and c) could indicate that the mycelia incubated at low temperature and pH were physiologically less active. This suggestion is supported by the observation that the proportion of 13C-Glc left in the medium when the mycelia were grown at low temperature and pH was higher than when they were grown under standard conditions. It is also possible to speculate that, besides a lower metabolic activity, the higher proportion of trehalose at the lower temperature might have been a response to a sudden decrease in the temperature. High concentrations of trehalose have been reported from

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FIG. 4. 13C-NMR spectra from mycelium of SNGT2-A grown under low temperature and pH conditions. FIGURE 4a and b as in FIG. 2. Abbreviations as in FIG. 2. alaalanine.

other fungi as a response to low temperatures (van Laere 1989, Mellor 1992). Large amounts of trehalose have been linked to membrane stabilization during dehydration/freezing stress in AM-fungi (Be card et al 1991). In our study, labeling in C6, C5, and C4 positions of trehalose indicates that some trehalose was synthesized via the gluconeogenesis pathway since it arises from labeled carbons from oxaloacetate (Martin et al 1985, Bago et al 1999). However, the ow of the isotopic carbon to these positions was lower than 10%, indicating that gluconeogenesis was not the main pathway. In all cases, we observed a weak isotopic scrambling between trehalose C1 and C6. This is similar to what was observed in P. tinctorius (Martin et al 1998), but in contrast to previous studies on C. geophilum and S. brunnea (Martin et al 1985, 1988). In the two latter studies, it was proposed that the mannitol cycle plays a key role in the cycling of glucose to form trehalose. Therefore it would be important to study the mannitol cycle en-

zymes in the vegetative mycelium of chanterelle, to determine to what extent this biochemical pathway in the carbon metabolism is active. In our study, it is likely that mannitol may have been produced at an early stage in higher proportions, and used later to build up trehalose and recycle glucose as it has been found in other ECM fungi (Martin et al 1985, 1998). Free amino acids accounted for a large proportion of the assimilated carbon. LBCT6 accumulated higher proportions of arginine than SNGT2-A, probably at the expense of glutamate exhaustion (Martin and Canet 1986). Glutamate was not detected in the LBCT6 spectrum, while in SNGT2-A a weak signal was observed. Similar 13C labeling patterns in glutamine C2, C3, and, C4 in both strains under the same culture conditions indicated randomization of the isotopic carbon via intermediates of the Krebs cycle (Martin and Canet 1986). Glutamate and its derivatives (arginine and glutamine) showed similar 13C labeling patterns in the spectrum in SNGT2-A cultivat-

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FIG. 5. 13C-NMR of exudated 13C compounds by the two strains of Cantharellus cibarius cultivated under two incubation conditions. * Grown under low temperature and pH conditions. Abbreviations as in FIG. 2.

ed at low pH and temperature, compared with the mycelia grown under standard conditions. Since the metabolism was slower at lower temperature, this was not observed in the strains incubated under standard

conditions. Enrichment of glutamate and glutamine suggests that the anaplerotic carboxylases are important (Straatsma and Bruinsma 1986, Martin et al 1998).

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FIG. 6. Proportion of 13C mannitol and trehalose from detectable 13C-NMR exudated from mycelia of two strains of Cantharellus cibarius grown under two different growing conditions, after 30 d of incubation. * Grown at 13 C and pH 4.5. The results come from pooled samples, therefore variation within each treatment was not calculated. Percentages illustrate signal intensity and not concentrations of the compounds. Abbreviations as in FIG. 1.

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cete Cenococcum geophilum monitored by 13C nuclear magnetic resonance. Physiol Veg 24:209218. , Ramstedt M, So derha ll K, Canet D. 1988. Carbohydrate and amino acid metabolism in the ectomycorrhizal ascomycete Sphaerosporella brunnea during glucose utilization. A 13C NMR study. Pl Physiol 86:935 940. . 1991. Nuclear magnetic resonance studies in ectomycorrhizal fungi. Techniques for the study of mycorrhiza. Methods Microbiol 23:121148. , Boifn V, Pfeffer PE. 1998. Carbohydrate and amino acid metabolism in the Eucalyptus globulusPisolithus tinctorius ectomycorrhiza during glucose utilization. Plant Physiol 118:627635. Mellor RB. 1992. Is trehalose a symbiotic determinant in symbioses between higher plants and microorganims? Symbiosis 12:113129. Ramstedt M, Martin F, So derha ll K. 1989. Mannitol metabolism in the ectomycorrhizal basidiomycete Piloderma croceum during glucose utilization. A 13C NMR study. Agric Ecosys Environ 28:409414. Rangel I, Danell E, Borowicz J, Martin F. 2000. Cantharellus cibarius: carbon and amino acid metabolism in relation to its fruit body-inhabiting uorescent Pseudomonas. In: Van Griensven LJLD, ed. Science and cultivation of edible fungi. Mushroom Science XV. Vol. 1. Rotterdam, Netherland: Balkema. p 8793. Smith SE, Read DJ. 1997. Mycorrhizal symbiosis. 2nd ed. San Diego, California: Academic Press. 605 p. Straatsma G, Konings RNH, Van Griensven LJLD. 1985. A strain collection of the mycorrhizal mushroom Cantharellus cibarius. Trans Br Mycol Soc 85:689697. , Van Griensven LJLD. 1986. Growth requirements of mycelial cultures of the mycorrhizal mushroom Cantharellus cibarius. Trans Br Mycol Soc 87(1):135141. , Bruinsma J. 1986. Carboxylated metabolic intermediates as nutritional factors in vegetative growth of the mycorrhizal mushroom Cantharellus cibarius Fr. J Plant Physiol 125:377381. Timonen S, Jorgensen K, Haahtela K, Sen R. 1998. Bacterial community structure at dened locations of the Pinus sylvestrisSuillus bovinus and Paxillus involutus mycorrhizospheres in dry forest humus and nursery peat. Can J Microbiol 44:499513. Van Laere A. 1989. Trehalose, reserve and/or stress metabolite? FEMS Microbiol Rev 63:201210. Wannet WJB, Op den Camp HJM, Wisselink HW, van der Drift C, Van Griensven LJLD, Vogels GD. 1998. Purication and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus. Biochim et Biophys Acta 1425:177188. , Aben EMJ, van der Drift C, Van Griensven LJLD, Vogels GD, Op den Camp HJM. 1999. Trehalose phosphorylase activity and carbohydrate levels during axenic fruiting in three Agaricus bisporus strains. Curr Microbiol 39:205210. , van der Drift C, Op den Camp HJM, Van Griensven LJLD. 2000. Trehalose and mannitol metabolism in Agaricus bisporus. In Van Griensven LJLD, ed. Science

We would like to thank Dr. Francis Martin (Equipe de Microbiologie Forestie ` re, INRA-Nancy) for his kind assistance in the 13C-NMR analyses of one of the strains. The project had support from the Swedish Council for Forestry and Agricultural Research, The Trygger Foundation and The National University of Mexico (UNAM/DGAPA).

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