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The use of ectomycorrhizal (ECM) fungi for afforestation, bioremediation, and timber production requires their maintenance over long periods under conditions that preserve their genetic, phenotypic, and physiological stability. Cryopreservation is nowadays considered as the most suitable method to maintain the phenotypic and genetic stability of a large number of filamentous fungi including the ECM fungi. Here, we compared the ability of eight ECM fungal isolates to colonize Pinus sylvestris roots and to transport inorganic phosphate (Pi) and NH4 + from the substrate to the plant after cryopreservation for 6 months at −130 °C or after storage at 4 °C. Overall, the mode of preservation had no significant effect on the colonization rates of P. sylvestris, the concentrations of ergosterol in the roots and substrate, and the uptake of Pi and NH4 +. Comparing the isolates, differences were sometimes observed with one or the other method of preservation. Suillus bovinus exhibited a reduced ability to form mycorrhizas and to take up Pi following cryopreservation, while one Suillus luteus isolate exhibited a decreased ability to take up NH4 +. Conversely, Hebeloma crustuliniforme, Laccaria bicolor, Paxillus involutus, and Pisolithus tinctorius exhibited a reduced ability to form mycorrhizas after storage at 4 °C, although this did not result in a reduced uptake of Pi and NH4 +. Cryopreservation appeared as a reliable method to maintain important phenotypic characteristics (i.e., root colonization and nutrient acquisition) of most of the ECM fungal isolates studied. For 50 % of the ECM fungal isolates, the colonization rate was even higher with the cultures cryopreserved at −130 °C as compared to those stored at 4 °C.

Scots pine (Pinus sylvestris L.) seedlings inoculated or not (NM) by a Zn-sensitive or a Zn-tolerant isolate of the ectomycorrhizal fungus Suillus bovinus (L. Fr.) Roussel were exposed to 0.1 or 150 muM Zn(2+) for 9 months. We hypothesized that inoculation with a Zn-tolerant S. bovinus isolate should result in added Zn resistance of the host plant. Plant and fungal growth as well as nutrient profiles and photosynthetic pigments in pine needles were quantified. In NM plants and in plants colonized by the Zn-sensitive isolate, plant growth, N, P, Mg and Fe assimilation were strongly inhibited under Zn stress and concurred with significantly reduced chlorophyll concentrations. In contrast, plants colonized by the Zn-tolerant isolate grew much better and remained physiologically healthier when exposed to elevated Zn. These results provide further evidence for the important role metal-adapted mycorrhizal fungi play as an effective biological barrier against metal toxicity in trees.

The ectomycorrhizal basidiomycete Suillus luteus (L.:Fr.), a typical pioneer species which associates with young pine trees colonizing disturbed sites, is a common root symbiont found at heavy metal contaminated sites. Three Cd-sensitive and three Cd-tolerant isolates of S. luteus, isolated respectively from non-polluted and a heavy metal-polluted site in Limburg (Belgium), were used for a transcriptomic analysis. We identified differentially expressed genes by cDNA-AFLP analysis. The possible roles of some of the encoded proteins in heavy metal (Cd) accumulation and tolerance are discussed. Despite the high conservation of coding sequences in S. luteus, a large intraspecific variation in the transcript profiles was observed. This variation was as large in Cd-tolerant as in sensitive isolates and may help this pioneer species to adapt to novel environments.

Zn pollution has triggered evolution for adaptive Zn tolerance in populations of Suilloid ectomycorrhizal fungi. The objectives of this study were to determine differential physiological responses that are linked to the Zn tolerance trait and to obtain more insight in the general mechanism responsible for the differential growth in Zn-enriched medium. Therefore, we identified intrinsic growth rates and element profiles in Zn-sensitive and Zn-tolerant genotypes. Isolates from Zn-polluted and unpolluted sites were exposed in vitro to increasing Zn(2+) stress. The Zn concentration which inhibits growth by 50% (EC(50)) was determined, and element (Zn, Fe, Mn, Cu, Mg, Ca and P) profiles in the mycelia were analysed. The intraspecific variation in growth rate and nutrient content of the in vitro grown mycelia is great and was not reduced in Zn-tolerant populations. The Zn resistance was not correlated to the intrinsic mycelial growth rate of the isolates or to the concentrations of the elements analysed, except for Zn. At low external Zn, Zn-resistant genotypes had lower Zn concentrations than sensitive isolates. At high external Zn, the differential Zn accumulation pattern between resistant and sensitive isolates became very prominent. Zn-exclusion mechanisms are most likely involved in the naturally selected adaptive Zn resistance. Other mechanisms of Zn detoxification such as sequestration of Zn on cell wall compounds or intracellular chelation and/or compartmentation are probably active but cannot explain the differential Zn sensitivity of the isolates.