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Summary • Nitrogen availability and colonization by mycorrhizal fungi may influence plant budgets of water, carbon, and carbon isotopes ( δ 13 C), but estimates of water use efficiency (WUE) derived from isotopic vs budgetary measurements are rarely compared. •W e assessed patterns of WUE, C allocation, and δ 13 C in cultures of nonmycorrhizal and ectomycorrhizal Pinus sylvestris at two N supply rates, 3% d − 1 and 5% d − 1 . • Mycorrhizal colonization increased δ 13 C at low N but not at high N. Relative to foliage, roots and mycorrhizal fungi were enriched in 13 C 1.5‰ and 3‰, respectively. 13 C fractionation during synthesis of transfer compounds of − 1.5‰ could account for this progressive enrichment. Increasing N availability increased instantaneous WUE by 7% but decreased budget-based WUE by 20%. WUE calculated isotopically was 10 times higher than budget-based WUE. Plants in our system therefore transpired most water without concurrent photosynthesis. •W e conclude that WUE depends on N concentration and not on the rate of N supply. The proportion of transpiration uncoupled from photosynthesis may largely control WUE in well-watered ecosystems, and this proportion depends on N concentration in the soil solution. Carbon isotopes only correlate with WUE above a critical concentration of available N. Thus, quantitatively interpreting δ 13 C patterns requires knowledge about coupling among C, N, and water in the plant‐mycorrhizal‐soil system.

Summary • Zn tolerance was investigated in populations of four ectomycorrhizal fungi: Suillus luteus, Suillus bovinus, Rhizopogon luteolus and Paxillus involutus. The fungi were collected in pioneer pine forests at 14 different locations, situated along a Zn pollution gradient. Genetic adaptation to Zn toxicity was previously presumed in a population of S. luteus. • Mycelial biomass production was assessed for 235 isolates exposed to increasing Zn2+ stress. EC50 concentrations were determined. • Adaptive Zn tolerance was found in the three species from the Suilloid clade and not in P. involutus. The Suilloid fungi collected within 5 km from a Zn smelter were highly Zn-tolerant, in contrast to isolates collected at least 15 km away from a pollution source. Mixed populations with tolerant and sensitive S. luteus isolates were found in a transition zone, between 5 and 15 km from the Zn smelters. • The severe Zn pollution in the surroundings of the Zn smelters has clearly triggered the evolution of an increased Zn tolerance in the pioneer Suilloid fungi. With increasing distances from the Zn smelters, the frequency of Zn-tolerant genotypes decreases.

Summary • Here we investigated zinc tolerance of ectomycorrhizal Scots pine (Pinus sylvestris) seedlings. An ectomycorrhizal genotype of Suillus bovinus, collected from a Zn-contaminated site and showing adaptive Zn tolerance in vitro, was compared with a nonadapted isolate from a nonpolluted area. • A dose–response experiment was performed. Dynamics of plant and fungal development, and phosphate and ammonium uptake capacity, were assessed under increasing Zn stress. Effects of Zn on transpiration, nutrient content and Zn accumulation were analysed. • Significant Zn–inoculation interaction effects were observed for several responses measured, including uptake rates of phosphate and ammonium; phosphorus, iron and Zn content in shoots; transpiration; biomass of external mycelia; and fungal biomass in roots. • The Zn-tolerant S. bovinus genotype was particularly efficient in protecting pines from Zn stress. The growth of a Zn-sensitive genotype from a normal wild-type population was inhibited at high Zn concentrations, and this isolate could not sustain the pines’ acquisition of nutrients. This study shows that well adapted microbial root symbionts are a major component of the survival strategy of trees that colonize contaminated soils.

Twenty-one isolates of the ectomycorrhizal fungus Suillus luteus were screened for their tolerance to the heavy metals Zn, Cd, Cu and Ni, measured as inhibition of radial growth and biomass production. Two populations from even-aged pine stands were investigated: 10 isolates were obtained from an area polluted with high levels of Zn, Cd and Cu, and 11 isolates were obtained from a control population located in a nearby unpolluted area. RFLP patterns of the internal transcribed spacer region of the isolates confirmed the morphological identification of the carpophores. All isolates were maintained on basic medium without elevated metals to avoid phenotypically acquired metal tolerance. The in vitro Zn and Cd tolerance of the S. luteus isolates from the polluted habitat were significantly higher than the tolerances measured in the isolates from the nonpolluted site. This observation suggests that the elevated soil metal concentrations might be responsible for the evolution of adaptive Zn and Cd tolerance. Tolerance was maintained in an isolate not exposed to elevated metals for 3 yr. The two S. luteus populations did not differ in tolerance to Cu and Ni. The mechanisms for the adaptive Zn and Cd tolerance are not identical as there was no correlation between response to the two metals; the most Zn-tolerant isolate was the most sensitive for Cd in the metal-tolerant population. Zinc did not accumulate in basidiocarp tissue, whereas Cd levels in basidiocarps were significantly higher in the population on the polluted site. Inter-simple sequence-repeat fingerprints showed that 90% of the isolates were from different individuals. The genetic variation in the population from the unpolluted site was considerably larger than that observed at the polluted site.

SUMMARY Short-term phosphate uptake rates were measured on intact ectomycorrhizal and non-mycorrhizal Pinits sylvestris seedlings using a new, non-destructive method. Uptake was quantified in semihydroponics from the depletion of Pi in a nutrient solution percolating through plant containers. Plants were grown for 1 or 2 months after inoculation at a low relative nutrient addition rate of 3 % d-1 and under P limitation. Four ectomycorrhizal fungi were studied: Paxillus involutus, Suillus lhteus, Suillus bovinuls and Thelephora terrestris. The Pi-uptake capacity of mycorrhizal plants increased sharply in the month after inoculation. The increase was dependent on the development of the mycobionts. A positive correlation was found between the Pi-uptake rates of the seedlings and the active fungal biomass in the substrate as measured by the ergosterol assay. The highest Pi-uptake rates were found in seedlings associated with fungi producing abundant external mycelia. At an external Pi concentration of 10 jM, mycorrhizal seedlings reached uptake rates that were 2.5 (T. terrestris) to 8.7 (P. involutus) times higher than those of non-mycorrhizal plants. The increased uptake rates did not result in an increased transfer of nutrients to the plant tissues. Nutrient depletion was ultimately similar between mycorrhizal and non-mycorrhizal plants in the semihydroponic system. Net P, absorption followed Michaelis-Menten kinetics: uptake rates declined with decreasing Pi concentrations in the nutrient solution. This reduction was most pronounced in nonmycorrhizal seedlings and plants colonized by T. terrestris. The results confirm that there is considerable heterogeneity in affinity for P, uptake among the different mycobionts. It is concluded that the external mycelia of ectomycorrhizal fungi strongly influence the Pi-uptake capacity of the pine seedlings, and that some mycobionts are well equipped to compete with other soil microorganisms for P, present at low concentrations in soil solution.

summary The decomposition and the nitrogen and phosphorus mineralization of fresh beech (Fagus sylvatica L.) leaf litter are described. Leaves were buried for up to 6 months in plant containers in which Scots pine (Pinus sylvestris L.) seedlings were cultivated at a low rate of nutrient addition. The saprotrophic abilities of three ectomycorrhizal fungi, Thelephora terrestris Ehrh.: Fr., Suillus bovinus (L.: Fr.) O. Kuntze and Paxillus involutes (Batsch: Fr) Fr., were compared with the degradation caused by the litter-decomposing basidiomycete, Lepista nuda (Bull.: Fr.) Cooke. Uninoculated leaves were included as controls. The investigation was performed at two different pH values since substrate pH is supposed to have an effect on the activities of extracellular enzymes of ectomycorrhizal fungi. The enzyme expression might also be largely influenced by the substrate they colonised. The mycorrhizal fungi caused only a low decomposition rate of the litter compared with that of L. nuda, and nitrogen was released only by L. nuda. Leaves colonized by mycorrhizal fungi showed no net release of nitrogen; on the contrary, a small accumulation of N in the litter was observed. It therefore seems likely that the ectomycorrhizal fungi studied do not have the ability to decompose efficiently the lignocellulose matrix of the relatively recalcitrant beech leaf litter. The degradation of this matrix seems to be essential for the fungi to gain access to the leaf nitrogen pool of fresh beech litter. A direct release of nitrogen from organic compounds by ectomycorrhizal fungi seems therefore to be confined to the older litter layers. The beech leaf litter contained an important fraction of easily mineralizable phosphorus. P was not a growth limiting factor m the cultivation system, and could therefore accumulate in the leaf litter colonized by the ectomycorrhizal mycelium.

SUMMARY The toxicity of a sublethal cadmium concentration to ectomycorrhizal Pinus sylvestris L. seedlings was studied. Nine mycorrhizal strains, collected from a Zn- and Cd-polluted soil or from an unpolluted area, were compared for their ability to increase Cd tolerance in their host plant. Plants were cultivated in a semi-hydroponic system that allowed visual observation of the root systems. The degree of soil colonization as well as the density of the extramatrical mycelium was determined. The water use of the host plants was carefully monitored during the treatment period. Cadmium concentrations were determined in all plant parts. Although Cd addition resulted in decreases in transpiration in the non-mycorrhizal plants, disturbances in water relations could be more or less pronounced in the mycorrhizal pines. The treatment strongly influenced the growth of the fungi in the substrate: several mycobionts hardly survived in the polluted substrate. Fungi producing a large mycelial biomass showed the greatest effect in overcoming toxicity. Some s trains were even able to increase their turnover growth when treated with cadmium. In a dense extramatrical mycelium the potential for Cd retention increases, while the individual hyphae are exposed to a lower Cd concentration than in a sparse mycelium. A protective effect against cadmium toxicity in the host was observed with all mycobionts, since Cd uptake was highest in the nonmycorrhizal seedlings. The treatment had no effect on the growth of the seedlings or on the concentrations of the other cations, probably due to the relatively low toxicity and the relatively short observation period. The results are discussed in relation to the in vitro Cd tolerance of the fungi.

SUMMARY Nine ectomycorrhizal fungi have been studied for their effect on the growth of Pinus sylvestris L. seedlings. The plants were kept growing for six months in root chambers with a changeable volume. All seedlings were cultivated under conditions of low substrate nutrient concentrations and they became strongly infected within a short time. We demonstrated the existence of an important negative correlation between the extent of the fungal development and the growth of the host plants. In most studies this reduced growth is attributed to an energy drain by the fungus. Fungi producing large amounts of fungal tissue are more energy consuming than species which only develop a sparse mycelium. However we suggest that also nitrogen acquisition by the plants might be altered by the fungus. Certain fungi probably retain a considerable amount of nitrogen for their own growth, thus reducing the amount transported to the host plant. A decreased N transport finally results in a slower growth. In this study the extramatrical mycelium in the rooting substrate was determined by wet oxidation and by nitrogen determination. This component of the fungal biomass showed the biggest variation among the different mycobiont species. The results are discussed in relation to the culture technique employed.