Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC6(3).

Growth and organelle morphology in the wood rotting basidiomycete fungus Phanerochaete velutina were examined in Petri dishes, on agar-coated slides, and in submerged cultures, using DIC, fluorescence and four-dimensional (4-D; x,y,z,t) confocal microscopy, with several fluorescent probes. Phanerochaete is ideal for this work because of its fast growth, robustness, and use in a wide range of other studies. The probe carboxy-DFFDA, widely used for labelling vacuoles, has no effect either on hyphal tip extension or colony growth at the concentrations usually applied in labelling experiments. Carboxy-DFFDA labels the vacuoles and these form a tubular reticulum in hyphal tip cells. The probe also labels extremely small vesicles (punctate fluorescence) in the apex of tip cells, the Spitzenkörper, and short tubules that undergo sequences of characteristic movements and transformations to produce various morphologies, including ring-like structures. Their location and behaviour suggest that they are a distinct group of structures, possibly a subset of vacuoles, but as yet to be fully identified. Regular incursions of tubules extending from these structures and from the vacuolar reticulum into the apical dome indicate the potential for delivery of material to the apex via tubules as well as vesicles. Such structures are potential candidates for delivering chitin synthases to the apex. Spitzenkörper behaviour has been followed as hyphal tips with linear growth encounter obstacle hyphae and, as the hydrolysis product of carboxy-DFFDA only accumulates in membrane-enclosed compartments, it can be inferred that the labelled structures represent the Spitzenkörper vesicle cloud. Mitochondria also form a reticular continuum of branched tubules in growing hyphal tips, and dual localisation with DiOC6(3) and CMAC allows this to be distinguished from the vacuolar reticulum. Like vacuolar tubules, mitochondrial tubules also span the septa, indicating that they may also be a conduit for intercellular transport.

Armillaria luteobubalina mycelium develops air pores that conduct oxygen to rhizomorph clusters.

Armillaria luteobubalina produces air pores in culture. They consist of two parts: a basal region of tissue elevated to form a mound covered with a rind continuous with that of the colony, but perforated; and an apical region of long parallel hyphae, cemented together by scattered patches of extracellular material. This forms a hydrophobic structure that is elevated above the general level of the mycelial crust and does not easily become waterlogged. Air pores develop near the inoculum plug shortly after inoculation, arising directly from the mycelium, and rhizomorphs are initiated from them. The air pore contains a complex system of gas space connecting the atmosphere with the central canal of each rhizomorph. The tissue beneath the melanised colony crust also contains gas space, especially near air pores. This is also connected with the gas space of each rhizomorph and of each air pore. Measurements with oxygen electrodes show that air pores and their associated rhizomorphs conduct oxygen. The average oxygen conductance of a group of air pores with associated rhizomorphs, within agar blocks, but with rhizomorph apices cut off, was about 700 x 10(-12) m3s(-1), equivalent to about 200 x 10(-12) m3s(-1) for each air-pore. We conclude that the air pores conduct oxygen into the gas space below the pigmented mycelium of the colony, where the rhizomorphs - which also conduct oxygen - originate. A. luteobubalina thus has a complex aerating system which allows efficient diffusion of oxygen into rhizomorphs, and this is likely to facilitate extension of inoculum into low-oxygen environments.

Phosphorus effects on metabolic processes in monoxenic arbuscular mycorrhiza cultures.

The influence of external phosphorus (P) on carbon (C) allocation and metabolism as well as processes related to P metabolism was studied in monoxenic arbuscular mycorrhiza cultures of carrot (Daucus carota). Fungal hyphae of Glomus intraradices proliferated from the solid minimal medium containing the colonized roots into C-free liquid minimal medium with different P treatments. The fungus formed around three times higher biomass in P-free liquid medium than in medium with 2.5 mM inorganic P (high-P). Mycelium in the second experiment was harvested at an earlier growth stage to study metabolic processes when the mycelium was actively growing. P treatment influenced the root P content and [(13)C]glucose administered to the roots 7 d before harvest gave a negative correlation between root P content and (13)C enrichment in arbuscular mycorrhiza fungal storage lipids in the extraradical hyphae. Eighteen percent of the enriched (13)C in extraradical hyphae was recovered in the fatty acid 16:1omega5 from neutral lipids. Polyphosphate accumulated in hyphae even in P-free medium. No influence of P treatment on fungal acid phosphatase activity was observed, whereas the proportion of alkaline-phosphatase-active hyphae was highest in high-P medium. We demonstrated the presence of a motile tubular vacuolar system in G. intraradices. This system was rarely seen in hyphae subjected to the highest P treatment. We concluded that the direct responses of the extraradical hyphae to the P concentration in the medium are limited. The effects found in hyphae seemed instead to be related to increased availability of P to the host root.

Apoplastic and symplastic pathways in the leaf of the grey mangrove Avicennia marina (Forsk.) Vierh.

Pathways for water and salts were traced from the vein endings to their sites of exit in leaves of Avicennia marina (Forsk.) Vierh. Investigations were conducted to determine whether the water and solutes separate at any point in the transpiration stream, and to identify their subsequent pathways. Leaves were placed with their cut petioles in solution containing one of two fluorochromes chosen to trace the transpiration stream. The apoplastic tracer was B-hydroxypyrene-1, 3, 6-trisulphonic acid (PTS), and the symplastic tracer was riboflavin buffered to pH 6.8. Leaf pieces were freeze-substituted with special care to keep the specimens dry to prevent redistribution of the water-soluble fluorochromes. Stages of the technique were sensitive to atmospheric moisture, so sections were cut in a room dried to 30% relative humidity, and they were stored in a sealed box containing desiccant. Riboflavin was never seen in the apoplast, and was inside the abaxtal (lower) glands within 30 min indicating a low resistance pathway through the symplast to the gland. The veins terminate in heavily pitted xylem vessels, and at the ends of these pits, the junction between the vein ending and the apoplast of the surrounding mesophyll, were high concentrations of PTS indicating regions of separation of the fluorochrome from water - places where the water crosses from the apoplast to the symplast. PTS diffused slowly in cell walls (measured diffusivity: 4.7 × 10-10 cm2 s-1 reaching the abaxial epidermis after 8 h, which was sufficient time for four complete changes of leaf water. It was concluded that the transpiration stream, probably containing the salt destined for secretion, moved symplastically from the xylem vein endings.