In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals.

We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid medium).

A Method for Radioactive Labelling of Hebeloma cylindrosporum to Study Plant-fungus Interactions.

In order to quantify P accumulation and P efflux in the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum, we supplied 32P to mycelia previously grown in vitro in liquid medium. The culture had four main steps that are 1) growing the mycelium on complete medium with P, 2) transfer the mycelia into new culture solution with or without P, 3) adding a solution containing 32P and 4) rinsing the mycelia before incubation with or without plant. The main point is to rinse very carefully the mycelia after 32P supply in order to avoid overestimation of 32P efflux into the medium.

Establishing a Symbiotic Interface between Cultured Ectomycorrhizal Fungi and Plants to Follow Fungal Phosphate Metabolism.

In ectomycorrhizal plants, the fungal cells colonize the roots of their host plant to create new organs called ectomycorrhizae. In these new organs, the fungal cells colonize the walls of the cortical cells, bathing in the same apoplasm as the plant cells in a space named the 'Hartig net', where exchanges between the two partners take place. Finally, the efficiency of ectomycorrhizal fungi to improve the phosphorus nutrition of their host plants will depend on the regulation of phosphate transfer from the fungal cells to plant cells in the Hartig net through as yet unknown mechanisms. In order to investigate these mechanisms, we developed an in vitro experimental device mimicking the common apoplasm of the ectomycorrhizae (the Hartig net) to study the phosphorus metabolism in the ectomycorrhizal fungus Hebeloma cylindrosporum when the fungal cells are associated or not with the plant cells of the host plant Pinus pinaster. This device can be used to monitor 32Phosphate efflux from the fungus previously incubated with 32P-orthophosphate.

The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31 P NMR spectroscopy study.

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32 P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32 P release compared with the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.

Dynamics of ectomycorrhizal mycelial growth and P transfer to the host plant in response to low and high soil P availability.

The aim of this work was to investigate the quantitative relationships between the rates of fungal soil exploration and their effects on plant growth and phosphorus (P) nutrition in soil with varying P availability. Ectomycorrhizal associations were established between Pinus pinaster and the basidiomycete Hebeloma cylindrosporum. Plants were grown for 4 and 6 months in mini-rhizoboxes filled with a 0.5 mm soil layer with two contrasting P levels (-P and +P soils), containing 3 or 50 mg of bicarbonate extractable P per kg of dry soil, respectively. Surface areas of the soil layers colonised by the roots and the hyphae were estimated using image analysis. High P availability decreased the rates of fungal soil colonisation, calculated as 0.92+/-0.19 cm(2) day(-1) plant(-1) in the -P soil and 0.42+/-0.1 cm(2) day(-1) plant(-1) in the +P soil over the 4-6 months period. Four-month old mycorrhizal plants accumulated lower amounts of biomass and total P than non-mycorrhizal plants, regardless the level of P availability. By contrast, 6-month old mycorrhizal plants were larger and contained more P than non-mycorrhizal plants, especially in the +P soil. However, mycorrhizal plants were always different from non-mycorrhizal P-deficient plants, which had an increased root surface and root P allocation. To explain these contradictory results, we propose that P accumulation by mycorrhizal plants derives mainly from fungal P uptake. The net P transfer from the fungus to the plant was estimated as 0.36 and 0.66 micromol of P per cm(2) of mycelium in -P and +P soil, respectively. Our data demonstrated that, despite the inhibitory effect of the high soil P availability on the rates of fungal soil colonisation, the ectomycorrhizal symbiosis was more efficient to improve host plant P nutrition in these conditions.