Experimental Approaches using Saprotrophic Cord forming Wood Decay Basidiomycetes to Investigate Effects of Carbon and Nitrogen Distribution on Cord Development and Translocation

2.2.1 Soil Nitrogen and Network Topology in Microcosms

Network topology has been found by mathematical network analysis to be adapted to exploit distant resources, and to survive interactions with other organisms that may delete parts of the network (Fricker et al., in press; Chapter 1). Network topology is species-specific in cord-forming wood decay fungi, reflecting short- and long-range foraging strategy, in other words, preference for small or large carbon resources (Boddy, 1999; Boddy and Jones, 2007). In the long-range foraging species P. velutina, individuals established in large dead logs extend corded mycelium into the surrounding soil, with diffuse fans of assimilating hyphae at their extremities (Boddy and Jones, 2007).

While it is the carbon resource that acts as the hub for extension growth, the extension rate and morphology of outgrowing mycelium are modified by the nitrogen environment. The ratio of carbon to nitrogen in the substratum determines the extent of cording of the mycelium, with nitrogen limitation acting as a cue for cord development in several species of saprotrophic cord-forming wood decay fungi. The morphogenetic effect of nitrogen content and C:N ratio have been investigated in axenic defined nutrient agar medium in P. velutina, C. puteana (common in European woodland) and S. lacrymans, an inhabitant of calcareous pine forest, from which the aggressive dry rot fungus of buildings has arisen (Kauserud et al., 2004). Interestingly, the switch to cord development occurred at similar carbon:nitrogen ratio in all three species. Factorial experiments demonstrated that carbon and nitrogen contents interacted and higher nitrogen was limiting at higher carbon. The effect on morphogenesis was different from that on biomass. Cord development was quantitatively related to nitrogen limitation, while biomass increased with both nitrogen and carbon.

Using mycelial C:N ratio to cue the development of nutrient conduits might be an adaptation to foraging under nitrogen limitation. The organism must prioritize carbon capture, as it cannot grow at all without an energy source. However, once carbon is sufficient, nitrogen is necessary for protein synthesis, for further growth and enzyme synthesis to exploit the wood carbon base. Mycelial topology is expected to be adapted to maximize the success of nitrogen scavenging in the nitrogen-limited environment. The developmental options (Boddy and Jones, 2007) are to toggle between diffuse, assimilatory mycelial growth and the development of hydrophobic (Olsson et al., 2002) mass flow channels resistant to attack, which extend the scavenging range by supplying carbon to distant foraging fronts to capture nitrogen over a wider area.

Subverting the link between amino acid uptake into the cell and developmental response can alter foraging network topology and prevent the organism from capturing fresh carbon resources (Dobson et al., 1993). This paramorphogenetic effect of a-aminoisobutyric acid (AIB, a non-metabolizable amino acid analogue) is exploited in its use to control the spread of dry rot from infected to uninfected timber elements in buildings. Mycelium charged with a high concentration of AIB

extends only very slowly, and remains as a localized symmetrical cushion of hyphae at the original carbon resource base. This morphology is typical of juvenile mycelium on a rich nutrient resource (Tlalka et al., 2007).

To investigate the effects of soil nitrogen pollution on woodland floor mycelial networks, we grew P. velutina, C. puteana and S. lacrymans from wood blocks, previously colonized for 2 or 7 months, over moist sand, with and without ammonium nitrate. Mycelia grown from the more decayed (less C-rich) blocks into nitrogen-free sand extended in mainly corded form, with diffuse assimilating mycelium limited to fans at the cord tips. Nitrogen (10 mg ammonium nitrate in 100 ml water added to 500 g sand) induced predominantly diffuse mycelium with few cords, initiated later. With nitrogen, the mycelium covered the plate rapidly and completely, compared with nitrogen-free sand where half the plate remained uncovered after 4 weeks (Figure 1). The concentration of nitrogen was

Figure 1 Balance between corded and diffuse mycelium growing from colonized wood blocks over sand. The 24 cm square dishes contained 500 g sand, to which was added either 100 ml deionized water or 10 mg of ammonium nitrate in 100ml deionized water. Mycelia were grown from beech wood blocks that had been colonized for 7 months, and were photographed at 2 and 4 weeks. (A) water, 2 weeks; (B) water, 4 weeks; (C) ammonium nitrate, 2 weeks; (D) ammonium nitrate, 4 weeks.

Figure 1 Balance between corded and diffuse mycelium growing from colonized wood blocks over sand. The 24 cm square dishes contained 500 g sand, to which was added either 100 ml deionized water or 10 mg of ammonium nitrate in 100ml deionized water. Mycelia were grown from beech wood blocks that had been colonized for 7 months, and were photographed at 2 and 4 weeks. (A) water, 2 weeks; (B) water, 4 weeks; (C) ammonium nitrate, 2 weeks; (D) ammonium nitrate, 4 weeks.

approximately 10-fold that recorded at the NERC ECN site at Wytham Wood, Oxfordshire. However, the results indicated a potential effect of eutrophication of woodland soils on saprotrophic fungal networks, which requires further investigation.

2.2.2 Nitrogen Limitation and Cord Initiation in Uniform Defined Media

Defined axenic culture is remote from ecological conditions, but it can parameterize a functional model to predict trigger points for N-induced switches in mycelial network topology. Using baseline data from defined media, carbon and nitrogen nutrient status of mycelium, and its likely effects on development in the natural habitat might be predicted. Figure 2 shows the relationship between nitrogen concentration in defined uniform nutrient agar media and biomass at 1 week of C. puteana mycelium, which was chosen for this experiment because it grows in culture with a relatively symmetrical geometry, compared with the highly variable morphology of P. velutina. Morphology was recorded as corded or uncorded. The curve of biomass against log nitrogen concentration was biphasic, suggesting a switch from high affinity, scavenging cell membrane transporters to a lower affinity system in the more nitrogen-rich environment. Interestingly, the discontinuity in the curve coincided with a clear switch, constant across three replicates, between corded and diffuse hyphae.

2.2.3 Photon Counting Scintillation Imaging of Changes in the Direction of Amino Acid Flow in Mycelium, Induced by Carbon Resource Capture

Nitrogen translocation into local carbon resources has been observed in the field (Frey et al., 2000) and at organism scale in microcosms (Watkinson et al., 1981). In

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Figure 2 Biomass increase with nitrogen supply, accompanied by suppression of cord development in Coniophora puteana at nitrogen content over 0.25 mg l_1. Basal medium, g l" sucrose, 20; KH2PO4, 1; MgSO4 • 7H2O, 0.5; FeSO4 • 7H2O, 0.01.

both cases, nitrogen import through mycelium enhanced the rate of cellulose decay. We do not know the time scale or sequence of cellular events leading from colonization of a localized insoluble cellulose resource to the induction of homeostatic amino acid translocation. By using photon counting scintillation imaging (PCSI) (Tlalka et al., 2002,2007; Bebber et al., 2007; Fricker et al., 2007), we can observe the effect of a fresh cellulose resource on the distribution of the free translocated amino acid pool in a mycelium, in real time. The microcosm used for the PCSI image (Figure 3) was designed to mimic the advancing edge of a mycelium in the forest floor, meeting and colonizing a fresh fragment of plant litter. A cellulose filter paper disc was placed near the advancing margin of a P. velutina mycelium in which the free amino acid pool had been labelled by prior

Figure 3 Photon counting scintillation image of a Phanerochaete velutina mycelium, to show distribution of translocated amino acid following colonization of a fresh cellulose resource. mycelium was grown over a scintillation screen from an agar inoculum disc to which 14C-labelled a-aminoisobutyric (AIB) acid at subtoxic concentration (10 p,l, 0.9 mmol) had been added. AIB is taken up and translocated but not metabolized, so remains unchanged and acts as a marker for movement of the free amino acid pool through the mycelium. The radiolabel, which was initially evenly distributed throughout the system, was translocated preferentially towards the new carbon resource.

Figure 3 Photon counting scintillation image of a Phanerochaete velutina mycelium, to show distribution of translocated amino acid following colonization of a fresh cellulose resource. mycelium was grown over a scintillation screen from an agar inoculum disc to which 14C-labelled a-aminoisobutyric (AIB) acid at subtoxic concentration (10 p,l, 0.9 mmol) had been added. AIB is taken up and translocated but not metabolized, so remains unchanged and acts as a marker for movement of the free amino acid pool through the mycelium. The radiolabel, which was initially evenly distributed throughout the system, was translocated preferentially towards the new carbon resource.

addition of 14C-AIB to the fungus. Within a few hours, the amino acid distribution throughout the colony changed, becoming asymmetrical as translocation was directed towards the added fresh resource.

The mechanism by which the organism perceives the local carbon resource is not known, but appears to be related to a nutrient signal because glass fibre discs, with similar hydrophilic physical characteristics to cellulose but no carbon content, did not induce this effect.

2.2.4 Capacity of Mycelium to Respond to Carbon and Nitrogen Resource Asymmetries by Translocation

The hypothesis that translocation operates to equilibrate separately acquired carbon and nitrogen for metabolic homeostasis was investigated by a series of experiments with the tractably symmetrical fungi C. puteana and S. lacrymans, both in Boletales, Coniophorales. Limiting nitrogen levels were established by testing each species over ranges of defined media. To mimic the effect of a network encountering a localized carbon or nutrient source and thus experiencing a patch of nutrient disequilibrium, cultures were grown on split plates with separate N and C sources.

When C. puteana mycelium, pre-grown on a permeable cellulose membrane over nitrogen-free agar medium, was transferred to a split plate with uniform high carbon as sucrose on one side but only nitrogen on the other, there was a striking differentiation of behaviour on each side of the plate. Mycelial extension, accompanied by cord development, accelerated on the N-limited side, apparently supplied with nitrogen by the cords that developed across the carbon-only medium. Extension ceased on the N-rich side, and cord development did not occur. However, biomass increased threefold, and metabolism appeared to alter, the mycelium releasing a dark brown pigment into the medium, presumably as a result of the onset of secondary metabolism.

In a short-term experiment, the pattern of equilibration of translocated amino acid between N-poor and N-rich sides of split plates was measured by scintillation counting of radiolabelled AIB added centrally to the inoculum. At 6 h there was much greater, but highly variable, allocation to the N-poor side, accompanied by a slight increase in biomass on that side. However, later, the distribution of AIB became much more uniform, and there was no consistent preferential allocation after 12 h. This could reflect an initial preferential allocation to carbon-rich cells, followed by futile circulation of the non-metabolized amino acid which remained in the mycelium and was not unloaded for biosynthesis.

2.2.5 Amino Acid Translocation between Compatible Individuals following Fusion

The discovery by Bebber (unpublished data) that fusion between two mycelial individuals is quickly followed by rapid amino acid flow from one to another suggests that equilibration of nitrogen throughout fused networks might be an important adaptive feature of foraging networks. This would enable formerly separate individuals to take advantage of others' foraging successes. Glass and Kaneko (2003) have drawn attention to the homeostatic and resource-sharing potential of fusion between individual networks, including the evolutionary significance of the limits placed by the vegetative incompatibility loci of fungi on the frequency of fusions between the individuals of a population.

In this context it is interesting that the cord-forming wood decay species S. lacrymans has been found to have unusually few compatibility types (Kauserud et al., 2006), so that chance-met individuals are likely to be able to fuse and share resources. Fungal vegetative compatibility genes that limit vegetative fusions between individuals in the field, by determining the number of vegetative compatibility types in the population of a species (Burnett, 2003), are believed to have evolved in response to selection against transmission of cytoplasmic viral infection when mycelia fuse (Glass and Kaneko, 2003). However, under resource heterogeneity, an individual that can link with a neighbour to extend joint network capacity acquiring distant limiting resources might be at a selective advantage. This is particularly likely in woodland where long-lived networks may frequently be temporarily fragmented under climatic stress, such as summer drought. Rapid reconnection to restore network capability might put the species at a competitive advantage.

Compatible and incompatible mycelia were grown together to compare the extent of amino acid translocation in fused and unfused networks (Figure 4). The incompatible dikaryons S7 and S16 of S. lacrymans, characterized as belonging to the A and D vegetative compatibility types, respectively (Kauserud et al., 2006), were grown in sand microcosms from paired colonized wood blocks. The inhibitory translocated amino acid AIB was locally applied to the margin of one individual when half-grown. The effect of AIB at very high concentration was to slow extension growth and induce a dense, regular margin without cords. In fused mycelia grown from paired blocks both colonized by S7, the effect of locally added amino acid took effect evenly throughout the system, while the incompatible mycelia grown from paired blocks colonized by S7 and S16 grew alongside each other but did not fuse, and inhibition was limited to the network containing AIB, which was subsequently engulfed by the uninhibited partner network (Figure 4).

2.2.6 Resource Sharing by Fusion may Enhance Biomass Production on Asymmetric Carbon and Nitrogen Resources

A short-term experiment suggested that homeostatic translocation may occur, and result in greater fitness, when compatible individuals with complementary nutrient requirements fuse. Compatible and incompatible pairings of S7 and S16 were set up on split plates. Most of the available carbon was on one side of the plate, and all the nitrogen on the other. Biomass on each side, and on control uniform media with either or both carbon and nitrogen, was recorded as a measure of fitness. Cords formed to bridge the gap between the sides of the plate in the compatible pairings only. After 1 week, connected mycelium on the carbon-only side weighed significantly more than unconnected mycelium (Figure 5). It would be interesting to investigate this effect further with realistic time scales and resources.

Figure 4 Transmission of inhibition throughout a fused mycelial system. Paired cultures of Serpula lacrymans grown from colonized pine (Pinus sylvestris) wood blocks placed side by side on sand, photographed 1 week after AIB addition. Pairs were either of Identical compatibility type, S7, which fused to form a single system (A and B), or of incompatible types, S7 and S16, which grew alongside each other but did not fuse (C and D). A 10% (w/v) aqueous solution of AIB, which at this concentration was a translocatable inhibitor of extension growth, was infiltrated into a cellulose filter paper disc placed across the advancing margin of one mycelium in 2 and 4; water alone was added in 1 and 3 incompatible mycelia from wood blocks colonized by S7 and S16 formed a barrage at the point of contact and grew beside each other at similar rates. The inhibition of extension caused by the presence of AIB in the translocated free amino acid pool was transmitted between compatible, but not incompatible, pairs of mycelia, indicating amino acid sharing on fusion between compatible individuals.

Figure 4 Transmission of inhibition throughout a fused mycelial system. Paired cultures of Serpula lacrymans grown from colonized pine (Pinus sylvestris) wood blocks placed side by side on sand, photographed 1 week after AIB addition. Pairs were either of Identical compatibility type, S7, which fused to form a single system (A and B), or of incompatible types, S7 and S16, which grew alongside each other but did not fuse (C and D). A 10% (w/v) aqueous solution of AIB, which at this concentration was a translocatable inhibitor of extension growth, was infiltrated into a cellulose filter paper disc placed across the advancing margin of one mycelium in 2 and 4; water alone was added in 1 and 3 incompatible mycelia from wood blocks colonized by S7 and S16 formed a barrage at the point of contact and grew beside each other at similar rates. The inhibition of extension caused by the presence of AIB in the translocated free amino acid pool was transmitted between compatible, but not incompatible, pairs of mycelia, indicating amino acid sharing on fusion between compatible individuals.

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