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- Materials and Methods
Cord-forming basidiomycete fungi form extensive mycelial networks that scavenge inorganic and organic nitrogen (N) efficiently from the soil environment (Boddy & Watkinson, 1995; Dighton, 1997). Their main source of carbon (C) for respiration or provision of C-skeletons for metabolism is wood, which has very low levels of N and other nutrients, such as phosphate (Boddy & Watkinson, 1995; Dighton, 1997). Thus considerable fluxes of N and phosphorus (P) must occur from remote assimilation sites or following autolysis of redundant mycelium to regions of high demand that arise during colonization and exploitation of new wood resource (Levi & Cowling, 1969; Venables & Watkinson, 1989; Cairney, 1992; Jennings, 1994; Boddy, 1999; Olsson, 2001). Resources are often scarce, ephemeral and patchy. As a result, the mycelial network develops as a highly plastic, interconnected functional unit that continuously senses and responds to nutritional cues in the environment and translates them into developmental responses (Rayner et al., 1995; Boddy, 1999; Ritz & Crawford, 1999; Olsson, 2001).
Most experimental data on nutrient transport in cord-forming mycelia is based on movement of radiolabelled phosphate (Cairney, 1992; Jennings, 1994; Boddy, 1999). There are fewer reports on N-translocation as there is no readily available radioactive N-isotope. Heavy nitrogen (15N) and mass spectroscopy have been used to study transport (Arnebrant et al., 1993), while stable N isotopes have proved useful in characterizing the metabolic pathways involved in mycorrhizal N metabolism using nuclear magnetic resonance (NMR) (Pfeffer et al., 2001), but have not yet been applied in imaging mode to resolve N transport. An alternative strategy has been to use a 14C-labelled amino-acid analogue, α-aminoisobutyric acid, (AIB) which is taken up but not metabolised (Ogilvie-Villa et al., 1981; Kim & Roon, 1982; Watkinson, 1984; Lilly et al., 1990; Olsson & Gray, 1998). Because AIB is not metabolized, the 14C-label faithfully reports the distribution of the amino acid analogue. Although early studies on 14C-AIB translocation used destructive sampling, dynamics of 14C-AIB movement were recently imaged in intact colonies of Schizophyllum commune using a β-scanner with spatial resolution in the order of a few millimetres and a theoretical sampling interval of about 1 h (Olsson & Gray, 1998).
To visualize N transport in mycelial networks with higher temporal and spatial resolution, we developed a novel noninvasive technique to track movement of 14C-AIB in mycelia grown over an inert scintillation screen using a photon-counting camera (Tlalka et al., 2002). Rapid, pulsatile movement of AIB was observed with a period of 14.5 h in growing, foraging hyphae and 11–12 h along developing cords. The oscillations were maintained for a considerable period in darkness at constant temperature.
Rhythmic phenomena are widespread in fungi and other simple eukaryotes, ranging from self-sustaining metabolic rhythms with periodicities of the order of minutes (Kippert & Hunt, 2000) to the archetypal circadian rhythms associated with sporulation in Neurospora (Dunlap, 1998; Ramsdale, 1999; Bell-Pedersen, 2000; Merrow et al., 2001). Circadian rhythms in amino-acid uptake have been described for yeast (Edmunds et al., 1979) and Synechococcus (Chen et al., 1991). While the period of the oscillations in 14C-AIB transport observed previously was clearly different from a canonical 24-h circadian rhythm, it is possible that the oscillations are tied into the output of an ultradian oscillator with a period of less than 24 hours (Dunlap, 1998; Kippert & Hunt, 2000).
In this paper we report on improvements in both the camera system and the scintillation screen that allow simultaneous measurement of signals from both assimilatory hyphae that grow on the agar inoculum and takeup nutrients from it, and distal foraging hyphae that extend over the surrounding nonnutrient area. With this more sensitive system we have tested: first, whether there are differences in the pulsatile behaviour within assimilatory and foraging hyphae; second whether the oscillations are coupled to pulses in growth; and third whether the rhythm observed is subservient to a central clock, and therefore reflects the output of that oscillator, or whether the oscillations are an intrinsic part of amino-acid uptake and translocation and may therefore yield information on the underlying control systems.