Following uptake of 32P-orthophosphate and 14C-aminoisobutyric acid (14C-AIB) the patterns of distribution of the isotopes through intact basidiomycete mycelia were non-destructively mapped at regular intervals using a β-scanner. Analysis of the results suggests that translocation of 32P and 14C-AIB through mycelia of Pleurotus ostreatus and Schizophyllum commune occurred along a restricted number of clearly defined, but macroscopically invisible, routes through the mycelium. In contrast to this, 32P added to mycelia of Coprinus cinereus remained immobilised at the addition point. Simultaneous acropetal and basipetal translocation of 32P and 14C-AIB was observed in different regions of colonies of P. ostreatus and S. commune. Translocation of label around the periphery of colonies strongly suggested the existence of anastomoses around the colony margin. Both 32P and 14C-AIB were initially immobilised at the addition point, from which each was subsequently translocated to other parts of the mycelium. The observed translocation of nutrients could not be explained by simple diffusion alone. The velocity of translocation and the complexity of the translocation pattern of 32P were greatest in mycelia of P. ostreatus, a hardwood decomposer, followed by S. commune, a wood and litter decomposer and parasite. Translocation through mycelia of C. cinereus, a coprophilus saprophyte, was very slow. This study provides the first detailed description of nutrient translocation through intact, entire fungal mycelia over time.
The nature and concentration of nutrients and other physicochemical factors vary greatly in space within the natural habitat of many fungi. This is especially true for fungi inhabiting soil. These fungi often produce large mycelia spanning a variety of microsites, each with different environmental conditions. Such fungi might be expected to be especially adapted for growth in heterogeneous environments. It is evident from a number of studies that wood and litter decomposing fungi are able to reallocate nutrients between different parts of their mycelium [1–4]. Extensive translocation of phosphorus has been shown to take place through mycelial cord systems of Phanerochaete velutina, Phallus impudicus and Hypholoma fasciculare grown in laboratory microcosms and in the field. In such systems, 32P-labelled phosphate was translocated 10 times more quickly through connective than through non-connective mycelium. Reallocation of phosphate within cord systems was dependent upon the potential of newly colonised, distant resource units as carbon sources, the decay state of those resource units and the temperature. The amount of phosphorus translocated between resource units was species-dependent, and 32P added to cord systems in the field was translocated for distances of up to 0.75 m, and was later detected in nearby decaying wood, leaf litter, and plants [2, 5–7]. Four main mechanisms have been suggested for the translocation of nutrients through fungal mycelia: diffusion ; diffusion aided by uptake in excess of local needs ; involvement of a contractile system ; and pressure-driven bulk flow [10, 11]. The last two mechanisms are active processes, requiring energy expenditure to drive the actual translocation process. It is important to note that the mechanisms are not mutually exclusive. It has been known for some time that acropetal and basipetal translocation of different nutrients can occur simultaneously in individual rhizomorphs of Armillaria mellea. In a previous study we showed that a nutrient, phosphorus, was translocated through a mycelium of Schizophyllum commune in a different and more active way than the non-nutrient caesium . This result supports the view that more than one of the translocation mechanisms described above may simultaneously be active in a mycelium.
In many previous studies, the translocation of nutrients through filamentous fungi has been measured along a one-dimensional structure [8, 12, 14]. In other studies, the rate of removal of a nutrient from or accumulation within a particular point or points within a mycelium was measured quantitatively [2, 15], or else translocation across a whole mycelium was measured qualitatively [15–17]. This inevitably gives an incomplete picture of the translocation processes occurring within a mycelium. Recent advances in the conceptualisation of fungal development at the organism level have resulted in mycelia being described as indeterminate, potentially infinitely expandable living systems. According to this paradigm, patterns of mycelial development are critically dependent upon the breakdown, maintenance and generation of connections allowing the reallocation of resources across the mycelium . It is, therefore, desirable to gain an improved understanding of nutrient translocation through filamentous fungi at the organism level, as translocation processes observed within isolated hyphae or cords, or at particular points within a mycelium, may not be representative of the behaviour of the mycelium as a whole.
The aim of this work was to study the translocation of phosphorus and amino acids through intact, whole mycelia of saprotrophic basidiomycete fungi. This was achieved by non-destructively recording the distribution of labelled phosphorus and [14C]2-aminoisobutyric acid (14C-AIB) through largely 2-dimensional agar cultures of wood and litter decomposing basidiomycetes through time. Three species were studied, in order to investigate whether fungal species differ in their ability to translocate nutrients. Similar experiments have been performed previously for 137Cs , a pollutant, but the present paper is the first to describe this for nutrients, in this case phosphorus and the amino acid analogue AIB. AIB was chosen as a marker for the amino acid pool because it is taken up by fungi and is translocated like other amino acids, without being metabolised .
2Materials and methods
2.1Origin and characteristics of fungal isolates
S. commune Fr.:Fr. ‘SC’ dikaryon and Pleurotus ostreatus (Jacquin: Fr.) Kummer ‘PO2’ dikaryon were provided by Dr. Paul Markham, King's College, University of London. Coprinus cinereus (Schaeffer: Fr.) S.F. Gray ‘Meathop’ dikaryon was provided by Dr. David Moore, School of Biological Sciences, University of Manchester. The fungi were maintained on malt extract agar (2% w/v malt extract broth+1.5% w/v agar No. 1, Oxoid).
Glass plates (3 mm thick standard glass used for window panes) were cut into 200×100 mm rectangles, sterilised by autoclaving and allowed to cool. Plates were placed in pairs into 240×240 mm sterile polystyrene plastic screening dishes (Nunc), and 110 ml malt extract agar was poured into each dish. This was sufficient agar to cover the glass plates to a depth of 1 mm. Each plate was inoculated centrally with an inverted single plugs (8 mm in diameter) cut from the edge of a stock cultures. Excess agar was shaved off each inoculum plug with a scalpel leaving only a mat of mycelium as the inoculum. This was done to prevent the agar from interfering with subsequent scanning in the β-scanner. Thereafter the plates were incubated at 20°C until the colony diameter had reached 90–100 mm.
2.3Labelling with 32P-orthophosphate and 14C-AIB
The glass plates carrying the fungal colonies were cut out from the agar. Label was then added into the agar at either the centre of the colonies, or at the colony edge, using a micro-pipette. Labelled phosphorus was added as 0.5 μl of an aqueous 32P solution (18.5 kBq; 55 fmol total P). Labelled AIB was added as 4 μl of a solution of 14C-AIB (29.6 kBq, 13 nmol total AIB) in 0.01 M HCl. The glass plates were then wrapped in polyethylene foil (Glad wrap) to prevent desiccation. The shape of the colony and the point of addition of radiolabel were carefully traced directly onto the foil by using a soft pen. This trace was also used to control that the radial growth rate during the incubation with the labelled compounds did not change compared to before labelling.
2.4Labelling with CaH32PO4
Labelled calcium phosphate was prepared by heating 400 μl phosphate solution (K2HPO4, 1.0 g l−1, pH 5.4), to which 20 μl H332PO4 solution (740 kBq, 2.2 pmol) had been added, together with 400 μl calcium solution (CaCl2·2H2O, 0.85 g l−1) at 50°C on a water bath until a precipitate of CaH32PO4 was formed. The precipitate was collected by dropwise addition of 80-μl aliquots of suspension onto discs of glass fibre filter paper (GF/A Whatman) 5.5 mm in diameter, placed on plain filter paper. The discs were then moved to new sheets of plain filter paper, and each was washed 10 times with 10 drops of distilled water. The resulting discs, loaded with CaH32PO4 crystals, were used for labelling fungal colonies. A single disc was added to each colony, either centrally or at the colony edge.
Plates which had not been inoculated with fungi were labelled in the same manner as the experimental plates described above. Where plates were labelled with 32P-orthophosphate or 14C-AIB, these controls provided a means of distinguishing between diffusive movement of label through the agar and translocation through fungal mycelia. Where plates were labelled with calcium 32P-phosphate, the controls were used to determine the degree of solubility of the calcium phosphate precipitate. Finally but most importantly, the controls were used to verify that the observed translocation was not an artefact of the experimental system.
2.6Determination of radiolabel distribution through mycelia
The distribution of radiolabel across experimental plates was carried out non-destructively using a β-scanner (Bioscan Imaging Scanner System 2000 equipped with Autochanger 1000). The scanner detects ionisation events resulting from radioactive decay by means of a 200 mm long position-sensitive anode wire. To improve resolution and to reduce scatter in the direction along the anode wire a metal window with a metal grid, a mechanical collimator, is fitted in front of the anode wire. The mechanical collimators can have different slit widths or different density of the metal grids. A high grid density gives higher resolution but lower sensitivity. Two-dimensional scanning is obtained by moving the Autochanger table in steps in a direction perpendicular to the anode wire. For the experiments described here, the glass plates were always oriented on the Autochanger table to move in steps along the shortest dimension (100 mm) of the glass plates. The position of decay events, and hence the distribution of radiolabel, was determined at a resolution of 0.78 mm along the length of the glass plates, and at a resolution of either 3 mm or 5 mm across the width of the glass plates depending upon the isotope used (see below).
The isotope 14C emits low-energy β-radiation which allows good spatial resolution of the origin of a decay event, but is prone to self-absorption of radiation by the sample. In contrast, 32P emits high-energy β-radiation which is less prone to self-absorption. This results in poorer spatial resolution than that obtained with a low energy isotope, but gives improved accuracy when quantifying the distribution of label below the surface of the mycelium or agar. Therefore, when measuring 14C-AIB, 30 Autochanger steps 3 mm apart were used together with a low resolution, high sensitivity mechanical collimator of 6 mm slit width for a counting time of 2 min for each step. For 32P, 19 Autochanger steps 5 mm apart were used together with a high resolution, low sensitivity mechanical collimator of 10 mm slit width for a counting time of 1 min for each step. Plates were maintained at a temperature 4–5°C below the 23–28°C in the scanner during scanning to prevent the formation of condensation on the inside of the foil . Plates were returned to the 20°C incubator immediately after scanning. The lower limits for detection of 32P and 14C-AIB by the β-scanner were 1.33×10−2 Bq mm-2 (3.84×10−5 fmol P mm−2) and 2.61×10−1 Bq mm−2 (114.7 fmol AIB mm−2) respectively.
2.7Replication and data processing
All of the experiments described above were carried out in duplicate. The data generated by the Bioscan system was exported as ascii files using a specially supplied ascii export program (Bioscan). Data manipulation and construction of two- and three-dimensional plots and subtraction plots was carried out using Excel (Microsoft). Diffusion coefficients of 32P and 14C-AIB were estimated from the control plates by two dimensional curve fitting of the standard equation for the distribution of substances in a two-dimensional diffusion to the experimental data, using the least squares method and employing Excel's Problem Solver.
Diffusion coefficients measured from the uninoculated control plates were used to calculate the distributions of radiolabel through the plates inoculated with fungi which would have arisen, had diffusion been the mechanism of translocation. The measured and calculated distributions of radiolabel were compared by construction of difference plots, where the distribution of label predicted by the diffusion model was subtracted from the measured distribution of label through fungal mycelia. A perfect fit of the laboratory data to the diffusion model would have given no difference in cpm for all points in X and Y. Positive values on such a difference plot indicate the presence of more label in a particular region of a colony than would be predicted on the basis of diffusion; negative values demonstrate the presence of less label in that part of the colony than would be predicted by the diffusion model.
The angle or direction of translocation ‘arms’ (see below) was determined from contour maps showing the distribution of radiolabel across each plate in plan view. Arms were located and marked on the map, and the angle of each arm from the positive y-axis measured to nearest 5° with protractor.
No macroscopic differentiation of fungal mycelia, such as cord formation or variation in hyphal density between regions of the mycelium of the same age, was observed in any of the experiments carried out. However, the three fungi reallocated the added nutrients in different ways. The different types of relocation pattern observed for the different fungi are summarised in Table 1 and Fig. 1.
Table 1. Patterns of nutrient translocation found in the experiments
Addition of label
Angle of ‘arms’
Duplicate plates (A and B) were scanned at approximately 0, 6, 24, 48 and 120–144 h after the addition of label to either the centre or the edge of the colony. The pattern of translocation between consecutive time intervals was categorised according to the key shown in Fig. 1. Where different translocation patterns were observed on the same plate at different times, all are listed. Where defined ‘arms’ of translocation were observed, the angle between each ‘arm’ and the positive y-axis was measured in a clockwise direction.
Symmetrical, 7.1, 7.2, 7.4
2, 3, 7.1, 8
7.1, −7.2, 7.3, −7.3
2, 6, 8
Symmetrical, 4, −4
Symmetrical, 2, 4, 6
−7.1, 7.1, 7.2
Symmetrical, 2, 6
Symmetrical, −7.1, 7.2, −7.3, 7.3
2, 6, 9
3.1Translocation of 32P and 14C-AIB through colonies labelled centrally
In mycelia of P. ostreatus both phosphorus and AIB were translocated out from the point of addition at the centre of the colony towards the edge of the colony along defined translocation ‘arms’ (Table 1; Figs. 1 and 2A,C). Both 32P and 14C-AIB were clearly detectable at the colony edge 24 h after addition of label. Difference plots, highlighting the change in distribution of radiotracer between 24 and 48 h (32P) or 48 h and 120 h (14C-AIB), show that label was lost from the central point of addition, and accumulated close to the edge of the mycelium (Fig. 3A,C). The pattern of translocation through S. commune was similar, but the rate of accumulation of label at the edge of the colony was slower and the ‘arms’ were less pronounced (Table 1; Figs. 1 and 4A,C). The velocity of the front of 32P through mycelia of C. cinereus was considerably slower than in either of the two other species studied. Translocation ‘arms’ were not apparent in C. cinereus, and radial symmetry in the distribution of radiolabel across the mycelium around the central point of addition was maintained (Table 1; Fig. 5A). In all fungi the velocity of the front of AIB was lower than that of phosphorus. It is not possible to be certain of the extent to which the difference in lower limits for detection of the two isotopes contributed to this difference.
Where translocation ‘arms’ were observed, the ‘arms’ on any individual plate were consistently evenly spaced (Table 1). Thus, on a plate with three ‘arms’ such as duplicate A of P. ostreatus labelled centrally with 14C-AIB, the angles of the arms clockwise from the positive y-axis were 5°, 120° and 240°. The angles between adjacent arms on this plate were therefore 115°, 120° and 125°. Similarly, where four ‘arms’ were observed they tended to be spaced at 90±10°. Where the angles between ‘arms’ were not even, they were consistent with those observed on a plate with one or two more ‘arms’. For example, duplicate B of S. commune labelled centrally with 32P had two ‘arms’ separated by 90°, which would give an even spacing on a plate with four ‘arms’.
3.2Translocation of 32P and 14C-AIB through colonies labelled at the edge
Labelled phosphorus or AIB added to the edge of mycelia of P. ostreatus was moved principally either towards the centre of the colony or around the periphery of the colony. There was also some translocation in the direction of growth of the leading edge of the mycelium (Table 1; Figs. 1 and 2B). The label translocated to the centre of the colony subsequently spread out from the centre, along ‘arms’ apparently similar to those observed when the label had been added centrally (Fig. 2B). Mycelia of S. commune reallocated label added to the edge of the colony in a pattern similar to that seen in P. ostreatus, but the velocity of the label front was lower and the patterns developed were less pronounced (Table 1; Fig. 4B,D). Label added to the edge of mycelia of C. cinereus was also reallocated towards the centre, around the periphery and towards the edge of the colony, but at a much lower velocity than in either of the other two fungi studied (Table 1; Figs. 1 and 5B). No label reached the centre of the colony before the end of the experiment. It is consequently not possible to be certain whether ‘arms’ of translocation would subsequently have developed.
3.3Comparison with a diffusion model
The observed movement of radiolabel through control plates not inoculated with fungi showed a good fit to that predicted by the standard diffusion equation (Fig. 6). The estimated diffusion coefficients were 4.0×10−6 cm2 s−1 and 3.6×10−6 cm2 s−1 for 32P and 14C-AIB respectively. Comparison of the measured distribution of 32P and 14C-AIB 24 h after addition to the centre of mycelia of P. ostreatus with that predicted by the diffusion model (Fig. 7) showed clearly that simple diffusion could not account for the transaction through the mycelium of either isotope. Label was accumulated more rapidly at the edge of the colony, and too much label remained at the centre of the colony. Translocation of both tracers added centrally to colonies of S. commune could again not be attributed to diffusion, for the same reasons as with P. ostreatus. Phosphorus added to C. cinereus remained in a symmetrical, Gaussian distribution, but and very little movement of label was detected (data not shown).
A direct comparison of the translocation of tracer added to the colony margin with the diffusion model was not made, due to the difficulty of deciding how to account for reflection of that part of the tracer within the mycelium at the boundary formed by the colony edge. It is clear, from the presence of translocation ‘arms’, that simple diffusion could not account for the translocation of either 32P or 14C-AIB observed in P. ostreatus and S. commune.
3.4Translocation of 32P added as insoluble calcium phosphate
Translocation of labelled insoluble calcium phosphate differed from that of labelled soluble phosphate in two ways. Firstly, reallocation of insoluble labelled phosphate was delayed by 24–48 h in comparison to soluble phosphate (data not shown). Secondly, the distribution of label through fungal mycelia after translocation had occurred deviated less from a symmetrical or Gaussian distribution where phosphate was added as an insoluble calcium salt, than where it was added as soluble orthophosphate (Table 1, Fig. 1). In control plates without fungi we unexpectedly found diffusion of the calcium phosphate label through the medium. The diffusion coefficient was approximately half of that estimated for 32P-orthophosphate.
The detection of both 32P and 14C-AIB at the edge of colonies of P. ostreatus and S. commune within 24 h of addition at the centre of the colony, 45 mm away, implies a velocity of the label front of at least 1.8 mm h−1. This is an order of magnitude faster than the reported velocity of translocation of 137Cs through intact mycelia of S. commune, Armillaria gallica and A. bulbosa[13, 21], and four times faster than the velocity of translocation of 86Rb, added as a tracer for potassium, through intact mycelia of Neurospora crassa. The contrast between the velocities of translocation of the tracers for nutrients reported here and the non-nutrient caesium confirms that fungi possess some mechanism for accelerated translocation of nutrients. It may be that, in a woodland soil environment, potassium is far less often limiting for growth of a fungus than phosphorus or amino acids. Consequently soil fungi may not have evolved such efficient mechanisms for translocation of potassium as for phosphorus and amino acids.
Velocities of translocation of 14C-aspartic acid and 32P-phosphate through cords of Serpula lacrimans in excess of 200 mm h−1, and of 32P-phosphate through rhizomorphs of A. mellea in excess of 20 mm h−1, have previously been reported [12, 15]. These studies were performed with specialised translocation structures of the differentiated fungi and were carried out in linear systems likely to enhance the rate of translocation observed through their design. This confirms the importance of carrying out studies of translocation on entire, intact mycelia.
The labelled nutrients were added in trace amounts (femtomolar and picomolar quantities), in a manner which does not allow the accurate calculation of the specific activities of either 32P or 14C-AIB relative to the total concentrations of phosphorus and amino acids present. Therefore the movements of tracer observed will largely reflect the translocation of amino acids and phosphorus already present in the mycelia, rather than translocation in response to the addition of the radiotracers. It must also be borne in mind when interpreting the results that the specific activity is unknown. Nevertheless it is clear that, particularly in P. ostreatus but also in S. commune, translocation of both phosphorus and amino acids took place along a restricted number of clearly defined but macroscopically invisible routes through the mycelium (Figs. 2–5). This was not the case with C. cinereus. Furthermore, simultaneous acropetal and basipetal movement of both 32P and 14C-AIB was observed in different regions of colonies of P. ostreatus and S. commune (Table 1). For example, in the case of replicate A of P. ostreatus labelled centrally with 32P, label was translocated acropetally along three ‘arms’ for the first 48 h after labelling. However, between 48 and 120 h after labelling, the direction of translocation in one of the ‘arms’ reversed, becoming basipetal, whilst translocation along the other two ‘arms’ remained acropetal. The translocation of nutrients observed could not be explained by simple diffusion alone, which may imply the presence of an active mechanism for translocation. Active translocation has most frequently been postulated as a method of nutrient translocation in wood-decomposing [1–4] and mycorrhizal basidiomycetes [3, 16, 17, 20], and arbuscular mycorrhizal fungi [21–23]. Evidence for active translocation in other fungi has also been published [4, 11, 24].
Comparison of the experimental data with the diffusion model suggested that label was immobilised at the point of addition to the mycelium. Similar observations were made by Clipson et al.  for the uptake of phosphorus by hyphal cords in the field. They suggested that the major fraction of phosphate absorbed by the cords was converted into an immobile form, not available for translocation. However, other studies have shown extensive translocation of phosphorus through mycelial cord systems in the field and in microcosms [2, 5–7]. In the present experiments, the immobilised label subsequently served as a pool of nutrient that decreased as the label accumulated in other parts of the mycelium (see Fig. 2).
The absence of macroscopic differentiation in the mycelia studied clearly does not preclude functional differentiation between hyphae, or differences at the microscopic level. The patterns of label distribution through mycelia of P. ostreatus and S. commune show striking similarities with those generated by a reaction-diffusion model proposed by Davidson et al.  to account for the large-scale properties of fungal mycelia. For this particular model, formation of peaks in the concentration of an activator (in that case biomass) near the colony margin was conditional upon the rate of replenishment of substrate exceeding the rate of decay of activator. The formation of clearly bounded, stable radial patterns, as a consequence of the amplification of small concentration gradients, has been documented in other biological systems . In the context of nutrient reallocation as described here, self-organisation of the mycelium to enable efficient translocation of nutrients could be achieved through modification of the degree of insulation of the boundaries to nutrient uptake and translocation (hyphal walls and septa), and through modification of the degree of overall resistance to flow through the mycelium . The regular spacing between clearly defined translocation ‘arms’, which was observed in mycelia of P. ostreatus and S. commune, may be due to self-organisation within the mycelia. The overall resistance to flow through the mycelium is increased by septation but reduced by anastomosis . The translocation of both 32P and 14C-AIB around the periphery of mycelia of P. ostreatus and S. commune, clearly seen when colonies were labelled at the edge (Table 1, Fig. 2B,D, Fig. 4B,D), strongly suggests the existence of anastomoses around the colony margin. Thus the patterns of nutrient reallocation observed here are consistent with the view of a mycelium as a self-organising, ‘communication network’ proposed by Rayner . Such a network will exhibit context-dependant macroscopic patterns of growth , which will include patterns of nutrient reallocation.
The pattern of nutrient translocation through S. commune was less rigidly bounded than that observed in P. ostreatus. Very little translocation was observed when label was added to mycelia of C. cinereus. The difference in translocation velocity and the complexity of the translocation pattern in the different species might reflect their adaptation to ecological niches varying in the heterogeneity with which carbon, nitrogen and phosphorus sources are distributed. Specifically, P. ostreatus is a primary decomposer of hardwood tree species; S. commune grows saprophytically on pine litter, and saprophytically and parasitically on woody substrates; and C. cinereus is a coprophilus saprophyte. Inter- and intra-specific differences in the ability of fungi to translocate carbon and mineral nutrients have been reported previously . Although caution must be exercised when applying data obtained in vitro to the field situation, the results reported above would support the hypothesis that inter-specific differences in the ability of fungi to translocate nutrients relate to the adaptation of particular fungal species or isolates to exploit a particular niche in soil ecosystems .
The most likely explanation for the unexpected diffusion of 32P added as calcium phosphate in the control plates is that the calcium phosphate was sparingly soluble in malt agar, which has a pH of 5.4±0.2. Even so, the rate of reallocation of labelled phosphate added to mycelia of P. ostreatus and S. commune was slower when phosphate was added in a sparingly soluble form than when it was added in a freely soluble form. Whether the observed differences in the pattern of reallocation of insoluble phosphate compared to soluble phosphate represent a major shift in the translocatory activity of the mycelium, or are simply an artefact of the slower rate of reallocation of insoluble phosphate, is uncertain. This merits further investigation, as there is little information on how fungi respond to the variety of chemical forms in which they encounter phosphorus and other nutrients in the field.
This work was supported by grants to S.O. from the Swedish Natural Research Council and The Swedish Agricultural and Forestry Research Council and was carried out at the Department of Microbial Ecology, Lund, Sweden. The work was also supported by a FEMS Fellowship for Young Scientists to S.N.G.