Imaging of Long-Distance α-Aminoisobutyric Acid Translocation Dynamics during Resource Capture by Serpula lacrymans

Culture. S. lacrymans (Wulf.) ex Schroet., isolate S7 (Bundesanstalt für Materialforschung und -prüfung, Berlin, Germany), was maintained in petri dishes on malt agar (4% Oxoid malt extract, 2% Oxoid no. 3 agar) at 22 ± 1°C in the dark in a temperature-controlled incubator (Gallenkamp, England).

Experimental microcosms.Autoclaved 1-cm³Pinus sylvestris sapwood blocks were colonized by S. lacrymans mycelium growing on malt agar for 30 to 60 days before transfer to a microcosm plate. Sand microcosm plates were 22- by 22-cm square Sterilin dishes containing 500 g of a 4:1 mixture of compacted black sand (Monro-Goundrey, United Kingdom) with acid-washed sand (VWR International Ltd., United Kingdom), evenly moistened with 80 ml deionized water. Black sand was necessary to make the white mycelium visible but was slightly hydrophobic, necessitating mixing with acid-washed sand to make a suitable growing surface for mycelium. Plates were inoculated with either one or two precolonized wood blocks and incubated in darkness at 22°C in polyethylene bags to maintain a high relative humidity. In some experiments, a new sterile wood block was added at a distance from the inoculated blocks to be colonized by the growing cultures during imaging.

Two microcosm arrangements were set up. The first microcosms (Fig. 2 and 3) were designed to test the effect and translocation of AIB following a single localized application at the margin of a complex network formed from the fusion of two clonal individuals grown from separate inoculum blocks. A single application, of either AIB or [¹⁴C]AIB, was made at a single point on the margin, as described below.

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FIG. 2.

Rapid and widespread inhibition of mycelial extension by 0.1 M AIB. Colonies of Serpula lacrymans were grown from paired adjacent precolonized wood inocula over nutrient-free moist sand in 22- by 22-cm square plastic dishes. At 10 days after inoculation, filter paper discs were placed at the mycelial margin and infiltrated with 300 μl of either 0.1 M AIB (A to C) or deionized water (D to F). The white dotted line (A to C) indicates the position of the mycelial margin at time zero. AIB treatment induced immediate global arrest of marginal extension and a change in the pattern of development, with tightly reticulate mycelial cords in panels A to C compared with radial cords in panels D to E.

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FIG. 3.

[¹⁴C]AIB translocation in a complex fungal network with successive basipetal and acropetal translocation. S. lacrymans colonies were grown from paired adjacent inocula, and after 10 days of growth, a filter paper disc placed on the margin was infiltrated with 10 μl 0.9 mM [¹⁴C]AIB with a specific activity of 2.11 GBq mmol⁻¹ (A). The plate was covered with a scintillation screen and imaged for 160 h. Translocations and accumulation of [¹⁴C]AIB in the fungal network are shown in panels B to E. Graph F shows the time course of the photon signal over the imaging period in selected regions of interest: L, [¹⁴C]AIB loading site; M1, mycelium connecting the loading site with the proximal inoculum block; M2, a representative area of mycelium at the advancing mycelial margin; B1 and B2, wood block inocula proximal and distal, respectively, to the [¹⁴C]AIB loading site. The photon signal was integrated over 6 h. The time following loading is given in pictures; the scale bar is 10 mm.

The second microcosm arrangement (Fig. 4) was designed to allow a mycelium preloaded with [¹⁴C]AIB as a marker to encounter and colonize one or two fresh wood blocks so that any ensuing changes in [¹⁴C]AIB distribution could be monitored. In these, [¹⁴C]AIB was applied directly to one of the inoculum blocks.

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FIG. 4.

Reallocation of [¹⁴C]AIB within a mycelial network induced by local colonization of a fresh wood resource. A 10-day-old mycelium grown from a colonized wood block over moist sand was loaded with 10 μl of 0.9 mM [¹⁴C]AIB applied directly to the top of the inoculum, and a sterile wood block (NW) was placed close to the mycelial margin (A). The whole plate, including the new wood block (but not the inoculum block, where the added [¹⁴C]AIB drop was still present), was then covered with a scintillation screen and imaged during further growth of mycelium and capture of the fresh wood resource (Fig. 3B to E). In all pictures, the photon signal was integrated over 12 h. Letters denote the inoculum loading site (I) and the new wood block (NW); time following loading is given in pictures; the scale bar is 10 mm. The relative redistribution of the label following localized resource capture is shown along the three chosen cords, indicated by dotted white lines (D). Panel F shows changes in distribution of [¹⁴C]AIB along cords 1, 2, and 3 during the imaging period. Cord 2 developed to connect the original inoculum with the fresh resource, which accumulated [¹⁴C]AIB (marked with a star), while the other two (cords 1 and 3) were not directly connected to it. In the x-t diagram, generated from images B to E in Video S2 in the supplemental material, the relative intensity of the signal elicited by the presence of [¹⁴C]AIB is shown as it changed along the length of each of the three selected cords simultaneously. The cords are represented by the three parallel columns, and time is shown vertically from top to bottom, representing the 390-h recording period of Video S2 in the supplemental material. In the columns corresponding to the three selected cords, the x dimension is shown oriented outwards from the inoculum block toward the mycelial margin.

Inhibitory AIB treatment.In experiments for inducing inhibition, 30 μmol (given as 300 μl) of 0.1 M AIB (Sigma, United Kingdom) solution was infiltrated into a filter paper disk placed in contact with the mycelium. This allowed the relatively large volumes of AIB solution used in inhibition experiments to be maintained in contact with the hyphae while preventing percolation into the underlying sand.

PCSI.Imaging of [¹⁴C]AIB was done using a high-resolution photon-counting camera system (HRPCS-3; Photek Inc. St. Leonards-on-Sea, United Kingdom) as described previously (33). [¹⁴C]AIB (0.9 mM) with a specific activity of 2.11 GBq mmol⁻¹ (Amersham, United Kingdom) was used. At this concentration, [¹⁴C]AIB did not inhibit the growth of the fungus, and the specific activity was low enough not to saturate the photon signal. For PCSI, where only 9 nmol of [¹⁴C]AIB was applied to the mycelium, [¹⁴C]AIB solution was introduced as a 10-μl drop either through the filter paper disc or straight onto a mycelium-covered wood block. After being loaded with [¹⁴C]AIB, the growing colonies were covered with BioMax TranScreen LE scintillation screen (GE Healthcare, United Kingdom). The scintillation screen was laid on the mycelium in close contact with it, taking care to avoid damage. Further mycelial growth continued in the confined plane between screen and sand. Rectangular openings were cut to accommodate wood blocks. Where wood block inocula were also imaged (Fig. 4), the rectangular screen cutout was placed on top of the block. The microcosms were then placed in the camera box and imaged continuously for up to 500 h with a 35-mm 1:1.4 Hama HR 0.5× lens (Nikon, Japan).

The resulting sequence file was then analyzed using IFS32 imaging software (Photek Inc., United Kingdom), allowing for user-defined integration with images integrated over 60 min for video display and 6 or 12 h for static pictures. The signals from [¹⁴C]AIB in hyphae, cords, and areas of the wood in contact with the screen were measured over time in the chosen regions. Liquid scintillation assays of [¹⁴C]AIB in extracts of sand at the end of the experiment showed some leakage from the mycelium into sand, where it would be undetectable by PCSI.

The signal was calibrated to link the intensity of pixels per unit area with the molarity of [¹⁴C]AIB. In brief, 10-μl drops of [¹⁴C]AIB containing the 50:50 dilutions of the compound (ranging from 1 to 18 pmol) were placed on the screen, allowed to dry, and imaged simultaneously next to the fungal microcosm under the same imaging conditions. The calibration curve showed good correlation between the molarity of [¹⁴C]AIB and the intensity of the photon signal. The advantage of estimating molar quantities from image intensity was that an estimate of rate can be inferred from video imaging (e.g., Fig. 3F). However, this method gives only a minimum value, based on the signal from [¹⁴C]AIB in close contact with the scintillation screen, and underestimates [¹⁴C]AIB in wood and sand substrates where the increased path length of β emissions and photons severely attenuates the signal.

The data for the different regions were transferred into Microsoft Office Excel and were plotted against time as pmol [¹⁴C]AIB mm⁻² of the selected region of the image. Changes in signal along selected cords were analyzed using Lucida 4.0 software (Kinetic Imaging, Liverpool, United Kingdom) to produce an x-t plot of intensity along the cord (x) against time (t). All images were assembled in Adobe Photoshop 7.0.

Quantifying [¹⁴C]AIB partitioning by liquid scintillation counting.Quantitative fractionation following PCSI was used to measure the proportion of [¹⁴C]AIB taken up that had been reallocated from the loading site into cords and fresh wood resources. After 6 weeks, different fractions of the microcosm plate were harvested. Fractions consisted of each of either two or three wood blocks, including the original inoculum and another fresh wood block(s); the mycelial cords growing over the sand between and beyond them; and samples of the sand mycelium (microcosms 1A to C and 2A to C) (Table 1). For the sand mycelium, 10 random 1-g samples of sand were collected. Each fraction was extracted in boiling 90% ethanol and [¹⁴C]AIB assayed by liquid scintillation counting of extracts. One-milliliter aliquots were pipetted directly into 4 ml OptiPhase Hisafe 3 scintillant (Fisher, United Kingdom), vortexed to mix, and counted with a Beckman LS1801 β spectrometer (Beckman Instruments Inc.). Partitioning of [¹⁴C]AIB between mycelial cords, wood blocks, and the sand mycelium was calculated in picomoles (Table 1). The [¹⁴C]AIB amount in the sand mycelium (as well as the total) is given only as an estimate due to the fact that data for the whole dish were extrapolated from the 10 random sand measurements. The aim of this analysis, complementary to the PCSI imaging, was to investigate and determine the partitioning of [¹⁴C]AIB between the inocula, the new wood blocks, and the remaining visible cords at the end of the experiment rather than to quantify the amount of the radiolabel in the sand. Three separate replicates were analyzed for each of the two types of microcosm described above.

Widespread fungal growth inhibition following local application of AIB.At high concentrations, AIB solution produced a rapid and widespread effect. A single local application of 30 μmol of AIB to the margin of a complex, fused mycelial network resulted in almost complete arrest of extension throughout the entire system, maintained throughout the 6-week experimental period (Fig. 2A to C). The more distant parts of the mycelium were equally affected, showing that inhibition was propagated irrespective of the relative positions of resources or growth sites. In controls (Fig. 2D and E), both colonies continued to extend throughout the 6-week period. Mycelial cord development was disrupted by AIB treatment, resulting in a tight reticulate network (Fig. 2C) in place of normal radial cord development (Fig. 2F).

Spatial dynamics of locally applied [¹⁴C]AIB through a complex network.At subtoxic concentrations, [¹⁴C]AIB did not affect mycelial extension. Following application of 9 nmol of [¹⁴C]AIB to a complex, fused mycelial network (Fig. 3A), [¹⁴C]AIB was immediately taken up from the filter paper and distributed throughout the system (see Video S1 in the supplemental material). Within 6 h, the signal increased in the mycelium (M1) and the first wood inoculum block (B1) (Fig. 3B). Accumulation of [¹⁴C]AIB increased in these regions in the following 30 h (Fig. 3C and F), with some spread through the neighboring cords and a significant decrease in the loading site. By 60 h, signal had become concentrated along the margin of the entire system (Fig. 3D). By the end of recording, at 160 h, [¹⁴C]AIB had accumulated in the second inoculum and the associated cords and nearby margin, while signal from the first inoculum was diminished (Fig. 3E and F).

Hourly integration of the signal in the area close to the loading site showed that [¹⁴C]AIB was initially imported at a rate of at least 0.024 pmol h⁻¹ (Fig. 3F, M1). The signal from this area subsequently declined at a similar rate, coinciding with redistribution of [¹⁴C]AIB throughout the network. Measurement of different regions of the colony also showed dynamic redistribution of [¹⁴C]AIB with transient accumulations in different areas, such as the growing margins (Fig. 3F, curve M2) and the wood block inocula (compare Fig. 3D and E with curves B1 and B2 in Fig. 3F).

AIB dynamics in response to carbon resource capture.To test whether AIB would be preferentially redistributed from the mycelium to a newly captured wood resource, the fungus was allowed to grow on sand toward a new wood block (Fig. 4A) before being loaded with [¹⁴C]AIB through the inoculum and imaged over time (Fig. 4B to E; also see Video S2 in the supplemental material).

Within 1 to 2 h after the inoculum was loaded, [¹⁴C]AIB appeared throughout the mycelium, rapidly reaching the margin (see Video S2 in the supplemental material). Detectable signal advanced along cords with a velocity of up to 13 mm h⁻¹ (cord 3). [¹⁴C]AIB then accumulated in cords and the growing margin for up to 100 h (Fig. 4C; also see Video S2 in the supplemental material) before being slowly removed from those sites (Fig. 4D; also see Video S2 in the supplemental material). Finally, by 390 h, a strong [¹⁴C]AIB signal had appeared at the surface of the newly colonized wood (Fig. 4E; also see Video S2 in the supplemental material).

The x-t analysis whose results are shown in Fig. 4F is based on PCSI from Video S2 in the supplemental material and shows the dynamics of [¹⁴C]AIB within a growing mycelium (photographed in Fig. 4A) as it captured and colonized a new wood block placed in the path of mycelial advance. Three cords were selected for analysis (Fig. 4D). These cords were chosen to compare the [¹⁴C]AIB dynamics of the region of the mycelium that became connected to the fresh resource and two other regions that did not. At 12 h, the x-t diagram was consistent with the [¹⁴C]AIB distribution shown in Fig. 4B. The selected cords all showed detectable signals, with the lowest signal in cord 2, which later connected the inoculum and fresh wood blocks. By 100 h, [¹⁴C]AIB had accumulated at the marginal mycelium supplied by all three cords, as shown by the darkened region on the right hand side of each column, and the root of each cord at the inoculum where [¹⁴C]AIB was loaded also appears darker than the main length of each cord, perhaps reflecting continued uptake of radiolabel.

At 230 h, the marginal mycelium had reached the fresh wood block and begun to colonize it. The changes in signal pattern that occurred in the period between 230 h and 390 h indicate the redistribution of [¹⁴C]AIB that followed this colonization event. The first noticeable change was in the marginal mycelium beyond the fresh block, seen as a new, darkened region on the right of the x-t column representing cord 2. This area shifted toward the right with time, showing that a marginal mycelium that had accumulated large amounts of [¹⁴C]AIB continued to grow radially away from the center and, indeed, appeared to accelerate, as shown by the diagonal slope of this region of the column, not seen in any of the cords at 100 h. Comparison of Fig. 4D and E shows that the mycelium extended several centimeters beyond the new block between 230 and 390 h.

At 390 h, there was a visible increase in signal from the new wood block itself, which started to appear at around 300 h, as seen on the x-t image. This almost certainly indicates localized accumulation of [¹⁴C]AIB rather than increased biomass of [¹⁴C]AIB-labeled mycelium, because there was room for only a thin layer of mycelium between the block and the overlaid screen. By this time, [¹⁴C]AIB had almost disappeared from cords 1 and 3. Taken together with the image shown in Fig. 4E and the analysis of [¹⁴C]AIB by destructive harvest, whose results are shown in Fig. 5, this indicates that [¹⁴C]AIB was withdrawn from throughout the mycelium immediately following colonization of the fresh wood block and simultaneously imported into wood and/or mycelium at the colonization site.

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FIG. 5.

AIB action in fungal microcosms. A schematic summary of the rates of uptake, accumulation in mycelium, and relocation to the mycelial margin and into a fresh wood resource based on the results is shown here.

To test the extent of [¹⁴C]AIB reallocation and accumulation during capture of a new carbon source, imaging by PCSI was combined with final liquid scintillation counting of microcosm fractions at the end of the 6-week experimental period. In Table 1, row 1 shows [¹⁴C]AIB partitioning in a microcosm where a wood block was added to a single colony (microcosms 1A to C) and row 2 shows a more realistic complex network resulting from fusion of adjacent mycelia (microcosms 2A to C). Table 1 gives the amount in picomoles of radiolabel recovered at the end of each experiment in each fraction for three replicate microcosms of each type.

In both types of microcosms, at harvest the freshly captured block contained significantly more [¹⁴C]AIB than the inoculum block, which had been the original site of [¹⁴C]AIB addition. Very little [¹⁴C]AIB was retained in the loaded inocula, suggesting a considerable translocation of the inhibitor to other places in the colony. This is confirmed by the presence of [¹⁴C]AIB in cords formed during the 6 weeks after loading.

In microcosm 1, the relative values of [¹⁴C]AIB distribution agreed remarkably well between replicate microcosms, with a threefold increase in fresh wood in comparison to the level in the inoculum block where [¹⁴C]AIB was applied. About 1.5 times more [¹⁴C]AIB was retained in the corded mycelium overlying the sand than in the new wood.

In the more complex network of microcosm 2, again [¹⁴C]AIB was preferentially allocated to the fresh wood block after transient accumulation in the first one (Table 1, row 2). In one microcosm (Fig. 3B), the advancing mycelium grew past the fresh block and did not colonize it. Here, most of the [¹⁴C]AIB held within cords and wood was allocated to the second block, which thus appeared to act as a transfer point from which [¹⁴C]AIB would probably be exported if a further resource were colonized. In both microcosm types, significant amounts of [¹⁴C]AIB were contained in the visible cords. The rest remained in the surrounding sand. It was not possible to separate uncorded diffuse mycelium from sand particles for analysis by scintillation counting. The [¹⁴C]AIB estimated to remain in the sand-plus-mycelium fraction includes that concentrated in the mycelial margins, as shown in Fig. 4, and any [¹⁴C]AIB released by the mycelial autolysis that accompanies remodeling of initial growth into the corded network (40).

The schematic diagram in Fig. 5 summarizes the results of the experiments described above to show the dynamics of AIB following uptake. Widespread translocation followed within 1 to 2 h, with selective distribution to mycelial margins and new resources through cords initiated by 12 h, accompanied by arrest of further extension, which was maintained throughout an experimental period of 6 weeks.