Plant and Soil 234: 109–117, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

109

Nitrogen limitation in mycorrhizal Norway spruce (Picea abies) seedlings induced mycelial foraging for ammonium: implications for Ca and Mg uptake Georg Jentschke1,2,3 , Douglas L. Godbold1 & Bettina Brandes2 1 School

of Agricultural and Forest Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, UK; Ecosystem Research Center, Institute of Forest Botany, University of Göttingen, Büsgenweg 2, D-37077 Göttingen, Germany. 3 Corresponding author∗ 2 Forest

Received 17 October 2000. Accepted in revised form 20 April 2001.

Key words: calcium, ectomycorrhiza, hyphal transport, magnesium, nitrogen nutrition, Paxillus involutus, Picea abies Abstract Although many studies support the importance of the external mycelium for nutrient acquisition of ectomycorrhizal plants, direct evidence for a significant contribution to host nitrogen nutrition is still scarce. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus (Batsch) Fr. in a sand culture system with two compartments separated by a 45-µm Nylon mesh. Hyphae, but not roots, can penetrate this net. Nutrient solutions were designed to limit seedling growth by nitrogen. Hyphal density in the hyphal compartment, host N status and shoot growth of mycorrhizal seedlings significantly increased in response to NH4 + addition to the hyphal compartment. Labeling the compartment only accessible to hyphae with 15 NH4 + showed that the increase in N uptake in the mycorrhizal seedlings was a result of hyphal N acquisition from the hyphal compartment. These results indicate that hyphae of P. involutus may actively forage into N-rich patches and improve host N status and growth. In the mycorrhizal seedlings, which received additional NH4 + via their external mycelium, the increase in NH4 + supply less negatively affected Ca and Mg uptake than in nonmycorrhizal seedlings, where the additional NH4 + was directly supplied to the roots. This was most likely due to the close link of NH4 + uptake and H+ extrusion, which, in the nonmycorrhizal seedlings, lead to a strong acidification in the root compartment, and subsequently reduced Ca and Mg uptake, whereas in the mycorrhizal seedlings the site of intensive NH4 + uptake and acidification was in the hyphal and not in the root compartment. Our data support the idea that the ectomycorrhizal mycelium connected to an N-deficient host may actively forage for N. The mycelium may also be important as a biological buffer system ameliorating negative influence of high NH4 + supply on cation uptake.

Introduction Mycorrhizas may significantly increase nutrient uptake of plants (Marschner and Dell, 1994; Smith and Read, 1997). In recent years it has become increasingly clear that the mycorrhizal mycelium emerging from the mycorrhizas into the soil is mainly responsible for this effect (Marschner and Dell, 1994; Smith and Read, 1997). This has predominantly been shown for arbuscular mycorrhizas (e.g., for N, Frey and ∗ Fax: +49-551-392705; E-mail: [email protected]

Schüepp, 1993; Johansen et al., 1992). For ectomycorrhizas, though a number of tracer studies have demonstrated the potential of ectomycorrhizal hyphae to increase N nutrition of their hosts (e.g., Ek et al., 1994, 1996; Finlay et al., 1988; Melin and Nilsson, 1952), direct evidence supporting a significant role of the external mycelium in mineral nutrition of forest tree seedlings is still scarce. Recently, using a compartmental culture technique, in which hyphal nutrient uptake could be isolated from root uptake, we have demonstrated that the external mycelium of Paxillus involutus contributed significantly to N and P nutri-

110 tion of N and P deficient Norway spruce seedlings (Brandes et al., 1998). As the seedlings were limited by both N and P, it, however, remained unclear from this experiment whether N or P limitation were the driving forces for mycelial foraging and translocation from patches of increased nutrient supply. Recent results by Perez-Moreno and Read (2000) suggest that Paxillus involutus may predominantly forage for P. However, hyphal foraging for N, independently of P, may be important in N limited acidic coniferous ecosystems to maximize N uptake. In this situation, a fungus responding to P and not N may be of limited benefit to the host plant. In many coniferous ecosystems, NH4 + is the dominant soil mineral nitrogen form (e.g., Harmer and Alexander, 1985; Persson and Wiren, 1995; Runge, 1983). As NH4 + uptake is linked to rhizosphere acidification (Marschner et al., 1991), and this may have negative effects on root base cation uptake, especially at elevated NH4 + supply (van Dijk and Roelofs, 1988; Gill and Reisenauer, 1993; Mehne-Jakobs and Gülpen, 1997), the question arises whether mycorrhizas may modify rhizosphere acidification when the external mycelium acquires most of the NH4 + . This may have major implications for the understanding of the impact of NH4 + deposition on forest tree mineral nutrition and growth. The aim of this study was to test the hypotheses (1) that hyphae of ectomycorrhizal plants solely deficient in N forage for nitrogen and translocate it to the host, thereby increasing plant growth, and (2) that altered patterns of rhizosphere acidification in mycorrhizal root systems reduce the sensitivity of mycorrhizal seedling cation uptake to high NH4 + supply.

constructed from PVC tubes with diameters of 10 and 15 cm forming a cylindrical inner compartment and a ring-shaped outer compartment. Four windows, which were covered with a 45-µm Nylon net, were cut into the inner tube. Hyphae, but not roots, can penetrate this net. The vessels were filled with acidwashed quartz sand and automatically irrigated with nutrient solution (composition see below). The nutrient solution was added at a rate of ca. 150 ml day−1 to the outer (root) compartment and ca. 30 ml day−1 to the inner (hyphal) compartment. The excess nutrient solution drained freely from the sand. The addition of a greater solution volume to the root compartment promoted mass flow from the plant to the hyphal compartment, and prevented mass flow in the opposite direction. Six seedlings were planted into the outer compartment of each vessel. Twelve vessels with non-mycorrhizal seedlings and eight vessels with seedlings colonized with P. involutus were set up. Vessels were kept in a climatic chamber at 20◦ C, a relative humidity of ca. 60% and continuous light with a photosynthetic photon flux density of 300 µmol m−2 s−1 . During the initial 4 weeks, nutrient solution (composition as in Marschner et al., 1996) was added only to the root compartment. The nutrient solution contained (µM): 300 NH4 NO3 , 50 Na2 SO4 , 100 K2 SO4 , 30 KH2 PO4 , 60 MgSO4 , 130 CaSO4 , 5 MnSO4 , 5 FeCl3 , 5 H3 BO3 , 0.1 Na2 MoO4 , 0.1 ZnSO4 , 0.1 CuSO4 , 120 HCl (pH 3.9). The vessels were randomized once a week to minimize variance in temperature and intensity of light. After 4 weeks, two plants were harvested from each of the growth vessels to determine the initial content of N in the plants. Experimental design

Materials and methods Plant culture and inoculation Seeds of Picea abies Karst. were surface sterilized with 20% (w/w) H2 O2 and germinated on water agar. When seedlings were 3 weeks old, they were transferred into a sterile perlite culture system. One half of them was inoculated with Paxillus involutus (Batsch) Fr. 533, isolated from a fruitbody collected at an acidified Norway spruce stand in northern Germany (Schlechte, 1986). The seedlings were grown for 9 weeks in perlite and then transferred to a twocompartment culture system described in detail by Brandes et al. (1998). Briefly, culture vessels were

After the initial harvest, irrigation of the hyphal compartments of all culture vessels was started. The experiment was a complete two-factorial design with factors mycorrhiza (nonmycorrhizal, with P. involutus) and N addition to the hyphal compartment (without N addition, with N addition). An additional treatment with nonmycorrhizal plants and N addition to both the root and hyphal compartment was included. This treatment will be referred to as ‘nonmycorrhizal, high N control’. Each treatment was replicated four times. All treatments had the following nutrient concentrations (µM) for both plant and hyphal compartment in common: 60/30 KH2 PO4 (plant/hyphal compartment), 200 K2 SO4 , 10/200 Na2 SO4 (plant/hyphal compartment), 60 MgSO4 , 130 CaSO4 , 10 MnSO4 , 7.8 Fe-

111 ethylenediamine-di(o-hydroxyphenylicacetic acid), 5 H3 BO3 , 0.1 Na2 MoO4 , 0.1 ZnSO4 , 0.1 CuSO4 . All input solutions had a pH of 3.9. Nitrogen was added solely as ammonium to exclude interactions between ammonium and nitrate uptake. Root compartment nutrient solutions had low N concentrations (150 µM NH4 Cl) to limit plant growth by N. The molar N:P ratio of the root compartment nutrient solution was 2.5, thus only one-fifth of the optimal ratio determined by Ingestad (1979). The NH4 + concentration used was significantly below long-term average values measured at the Solling site (northern Germany; Matzner et al., 1982). Nutrient solutions of the hyphal compartment contained 1.8 mM NH4 Cl or no N, according to the treatment. This relatively high N concentration was chosen to create a strong gradient in N availability between the root and hyphal compartment, which may mimic a situation in which additional soil N is available in N-rich patches. The NH4 Cl for the hyphal compartments was enriched in 15 N (10 atom%, Sigma) to trace plant N uptake from the hyphal compartment. The root compartments received ca. 22 µmol 14 N, and the hyphal compartments 0 or 50 µmol 14 N and 0 or 5 µmol 15 N per day according to the treatment. Total N input into the root compartments over the 10week experimental period was checked as described previously (Brandes et al., 1998). Briefly, aliquots of nutrient solutions were pooled, by individual vessels, over time. The pooled samples were analyzed for NH4 + , NO3 − and total N using standard autoanalyzer procedures (Cenco Instrumenten BV, Breda, The Netherlands). Nitrate was not detected in draining solutions during the experimental period, and concentrations of organic N [calculated as difference between total N and mineral N (NH4 + + NO3 − )] were negligible, indicating that microbial transformations of N were not significant in the system. For the nonmycorrhizal plants in the nonmycorrhizal, high N control, the amount of 14 NH4 + supplied to the root compartment was similar to the total N input (hyphal + root compartment) in treatments with N addition to the hyphal compartment. They received ca. 66 µmol 14 NH4 + per day at a concentration of 450 µM. In addition, the hyphal compartment was labelled with 15 N (10 atom%) as in the +NH4 + treatments. Harvest and analysis of the plant material After 10 weeks of treatment, plants were separated into needles, stems, and roots. The percentage of

mycorrhizal root tips was determined by counting a representative sample with the aid of a stereo microscope. All parts of the plants were dried to a constant weight at 70◦C and homogenized with a ball mill. The C and N content was determined with a C/N analyzer (Na1500, Carlo-Erba, Milan, Italy), atom% 15 N by mass spectrometry. The atom% 15 N excess was calculated by subtracting the atom% 15 N of nonmycorrhizal and mycorrhizal plants which had not received any 15 N label, (namely 0.380 for both types of plants) from the measured atom%. Shoot N and 15 N concentrations were calculated as weighted means of needle and stem concentrations. For determination of Ca and Mg, plant material was wet ashed using 65% (w/w) HNO3 in closed Teflon vessels under high pressure at 180◦C. Concentrations of Ca, Mg and P were determined by inductively coupled plasma atomic emission spectroscopy (Spectroflame, Spectro Analytical Instruments, Kleve, Germany). Hyphal density in the sand was determined by a direct counting method as described previously (Brandes et al., 1998). Statistical analysis was carried out using two-way analysis of variance (SAS Institute, Cary, NC, USA, 1987) with factors mycorrhiza (nonmycorrhizal, P. involutus) and N supply (without N, with N in hyphal compartment). Significance between means was tested using the Tukey test (SAS Institute). For the comparison of NH4 + fertilisation effects on nonmycorrhizal version mycorrhizal seedlings, treatments with P. involutus and with or without N addition to the hyphal compartment were compared with nonmycorrhizal seedlings that either received no additional N or received additional N in the root compartment (nonmycorrhizal, high N control). For the statistical analysis of nutrient contents, two-way analysis of variance with factors mycorrhiza (nonmycorrhizal, P. involutus) and N supply (low N, high N) was used. To increase homogeneity of variance if necessary, the data were log transformed before statistical analysis. Results Mycorrhizas and seedling dry weight By the end of the experiment short roots of seedlings inoculated with P. involutus were ca. 90% mycorrhizal. These figures were independent of NH4 + addition to the hyphal compartment. No mycorrhizal tips were found on noninoculated seedlings. Mycorrhizal seedlings had significantly higher shoot and

112

Figure 1. Effect of NH4 + addition to the hyphal compartment on dry weight of needles and roots of nonmycorrhizal and mycorrhizal (P. involutus) Norway spruce seedlings. Means (n=4) within graphs without common letters are significantly different (Tukey test, P≤0.05). Probability levels for ANOVA: ∗ P≤0.05; ∗∗ P≤0.01; ∗∗∗ P≤0.001; ∗∗∗∗ P≤0.0001. Myc, Mycorrhiza.

Figure 3. Concentration of 15 N (expressed as percentage 15 N of total N) in roots and shoots of nonmycorrhizal and mycorrhizal (P. involutus) Norway spruce seedlings after labeling the hyphal compartment in the culture system with 15 NH4 + (10 atom%) for 10 weeks. Nonmycorrhizal + 14 N: nonmycorrhizal, high N control (seedlings received additional 14 N in the root compartment). Broken line indicates 15 N abundance in nonmycorrhizal and mycorrhizal seedlings not treated with 15 N (0.38 atom%). Values are means of four replicate pots. Small bars indicate standard deviation. Values within graphs without common letters are significantly different (Tukey test, P≤0.05).

Nitrogen uptake and hyphal translocation

Figure 2. Nitrogen concentrations in roots and shoots of nonmycorrhizal and mycorrhizal (P. involutus) Norway spruce seedlings as affected by NH4 + added to the hyphal compartment. Means (n=4) within graphs without common letters are significantly different (Tukey test, P≤0.05). Probability levels for ANOVA: (∗ ) P≤0.1; ∗ P≤0.05; ∗∗∗ P≤0.001; ∗∗∗∗ P≤0.0001. Myc, Mycorrhiza.

root dry weight than nonmycorrhizal seedlings. Nitrogen added to the hyphal compartment significantly increased needle dry weight in mycorrhizal, but not in nonmycorrhizal seedlings (Fig. 1). ANOVA showed a significant Mycorrhiza × N supply interaction. For roots (Fig. 1) and stems (data not shown) interactions were not significant. Ammonium added to the root compartment of nonmycorrhizal seedlings increased both root and needle dry weight by 46 or 114%, respectively (P≤0.05). The seedlings of the nonmycorrhizal, high N control (1.7/1.7g root/needle dry weight per vessel) had approximately the same dry weight as the mycorrhizal seedlings with N added to the hyphal compartment (1.6/2.0 g root/needle dry weight per vessel).

Nitrogen addition to the hyphal compartment increased both the N concentration (Fig. 2) and content (Table 1) in the mycorrhizal but not in nonmycorrhizal seedlings. Nitrogen uptake during the N treatment period, therefore, was 2–4-fold greater in plants colonized with P. involutus and with N added to the hyphal compartment than in all other plants (Table 1). Nitrogen input to the root compartment during this period was only 22–23 mg per vessel. Nitrogen uptake by the mycorrhizal seedlings with N added to the hyphal compartment exceeded N input to the root compartment by almost a factor 2, whereas in all other treatments, N uptake was lower than N supply to the root compartment. As N transfer from the hyphal to the root compartment by diffusion or mass flow is low in our compartmental system (Brandes et al., 1998), the plant N content in excess of the root compartment supply most likely resulted from hyphal N acquisition. Based on calculations in Table 1, hyphal contribution to host N acquisition was estimated as a minimum of 43%. To confirm this assessment, we measured 15 N transfer from the hyphal compartment to the host plants. 15 N concentrations in shoots and roots were ca. 5-fold greater in mycorrhizal than in nonmycorrhizal seedlings (Fig. 3). Low 15 N concentrations in plants

113 Table 1. Initial and final N content and N uptake of nonmycorrhizal and mycorrhizal Picea abies seedlings grown in a two-compartment culture system with or without NH4 + addition to the hyphal compartment. Total N uptake was calculated as difference between final (week 10) and initial (week 0) seedling N content. Minimal N uptake from the hyphal compartment was calculated as the difference between total N uptake and N supply to the root compartment, assuming maximal N uptake from the root compartment to equal N supply to this compartment. Means (n=4) within columns without common letters are significantly different (Tukey test, P≤0.05) Treatment

N content (mg (vessel)−1 ) Initial

Nonmycorrhizal Control 7.6ab +NH4 + 6.5b Paxillus involutus Control 10.2a 8.0ab +NH4 +

Total N uptake (mg vessel−1 )

N supply to root compartment (mg vessel−1 )

17.8c 21.6c

10.2c 15.0bc

22.6a 23.4a

−12.4b −8.4b

29.2b 47.2a

19.0b 39.2a

23.1a 22.4a

−4.1b 16.8a

Final

Calculated minimal N uptake from hyphal compartment (mg vessel−1 )

of the nonmycorrhizal, high N control (‘Nonmyc. + 14 N’), which were the same size as the mycorrhizal seedlings, indicated that mass flow and diffusion in the rooting substrate did not confound measurements of hyphal15N translocation. Based on the 15 N data, the total amount of N derived from the hyphal compartment was ca. 15 times higher in mycorrhizal than in nonmycorrhizal seedlings of the nonmycorrhizal, high N control (Table 2). Hyphal contribution to total N uptake in mycorrhizal seedlings was estimated as 45% (Table 2). Needle phosphorus and potassium concentrations and N:P, N:K and N:Mg ratios Needle P concentations in nonmycorrhizal and mycorrhizal seedlings ranged between 65 and 72 µmol (g d. wt.)−1 or 54 and 58 µmol (g d. wt.)−1 at the final harvest, respectively, and were not affected by NH4 + addition to the hyphal compartment (P>0.05). Molar N:P ratios ranged between 6.6 and 7.0 in nonmycorrhizal seedlings. In mycorrhizal seedlings, addition of NH4 + to the hyphal compartment increased the N:P ratio from 7 to 10 (P≤0.05), still below the optimal proportion of 12 (Ingestad, 1979). Molar N:Mg ratios ranged between 9 and 22, clearly staying below the optimal value of 30 (Ingestad, 1979). Needle potassium concentrations ranged between 210 and 250 µmol (g d. wt.)−1 in all treatments. Molar N:K ratios were between 1.9 and 2.3 for all treatments and thus far below the optimal proportion of 5 determined by Ingestad (1979).

Figure 4. Effect of NH4 + addition to the hyphal compartment on hyphal length in the root and hyphal compartments of seedlings inoculated with P. involutus. Data were transformed into common logarithms before statistical analysis (one-way ANOVA). The figure shows backtransformed means of four replicates. ∗∗ Significant difference between control and +NH4 + plants (P≤0.01).

Hyphal length In nonmycorrhizal treatments, hyphal length in the sand was below 0.01 m (g sand)−1. In contrast, hyphal length in all mycorrhizal treatments was signific-

114 Table 2. 15 N uptake by nonmycorrhizal and mycorrhizal Picea abies seedlings from the hyphal compartment. Nonmycorrhizal and mycorrhizal (P. involutus) seedlings were grown in a two-compartment culture system with 15 NH4 + addition (10 atom% 15 N) to the hyphal compartment for 10 weeks. Nitrogen (14 N + 15 N) uptake from the hyphal compartment was calculated by dividing excess 15 N in the seedlings by the 15 N atomic excess of the labeling solution (9.62%). Means (n=4) within columns without common letters are significantly different (Tukey test, P≤0.05) Mycorrhiza

Nonmycorrhizal Nonmycorrhizal, high N control Paxillus involutus

Excess 15 N in seedlings (mg vessel−1 )

14 N + 15 N uptake

from hyphal compartment (mg vessel−1

Percentage of N uptake from hyphal compartmenta

0.05b 0.13b

0.5b 1.2b

4b 7b

1.85a

17.4a

45a

a Total N uptake (Table 1) = 100%.

antly (P≤0.05) 40–100-fold above background values (Fig. 4). Nitrogen addition to the hyphal compartment did not affect the hyphal length in the root compartment, but increased it in the hyphal compartment (Fig. 4). Ca and Mg concentrations in roots and needles as affected by NH4 + supply For the evaluation of NH4 + effects on Ca and Mg uptake of mycorrhizal and nonmycorrhizal seedlings, we compared mycorrhizal seedlings without or with NH4 + added to the hyphal compartment (low N, high N) with nonmycorrhizal seedlings that received control levels of NH4 + (low N) or additional NH4 + in the root compartment (high N). In spite of the different location of the N addition (root versus hyphal compartment), the effects on N tissue concentrations in both nonmycorrhizal and mycorrhizal seedlings were very similar (Table 3). ANOVA revealed that Mycorrhiza × N supply interactions were not significant for both root and needle concentrations, while NH4 + supply significantly increased N concentrations in both roots (F=41, P≤0.0001) and needles (F=29, P≤0.0001). Root N concentrations were significantly higher in seedlings colonized with P. involutus than in nonmycorrhizal seedlings (F=99, P≤0.0001). With the exception of root Ca, Ca and Mg concentrations in roots and needles showed significant Mycorrhiza × N supply interactions (Table 3). Increased NH4 + supply reduced root Mg concentrations in nonmycorrhizal seedlings by ca. 15%, but did not affect them in mycorrhizal seedlings. High-N mycor-

rhizal seedlings had ca. 30% higher root Mg concentrations than corresponding nonmycorrhizal seedlings. In needles, increasing NH4 + supply reduced Ca and Mg concentrations less in mycorrhizal than in nonmycorrhizal seedlings (Table 3). Additional NH4 + uptake reduced the pH in the root compartment much stronger in nonmycorrhizal than in mycorrhizal seedlings. ANOVA revealed a highly significant Mycorrhiza × N supply interaction (Table 3). In the hyphal compartment a reversed pattern was observed.

Discussion Seedling nutritional status In contrast to previous studies where seedlings were either limited by P (Jentschke et al., 2001) or both P and N (Brandes et al., 1998), the seedlings in this experiment were solely limited by N. This is evident from the strong growth responses of nonmycorrhizal and mycorrhizal seedlings to nitrogen added to the root or hyphal compartments, respectively. In addition, molar N:P ratios in needles of nonmycorrhizal and mycorrhizal seedlings were in all cases lower than the optimal ratio of 12 determined by Ingestad (1979). Molar N:K and N:Mg ratios were also clearly below optimal ratios determined by Ingestad (1979).

115 Table 3. Effect of NH4 + addition on concentrations of N, Ca, and Mg in needles and roots of non-mycorrhizal and mycorrhizal (Paxillus involutus) Norway spruce seedlings and on pH (week 10) in the draining solutions from root and hyphal compartments. The additional NH4 + was directly supplied to the roots of non-mycorrhizal seedlings and indirectly supplied to mycorrhizal seedlings via the hyphal compartment. The pH of all input solutions was 3.9. Ca and Mg concentrations were log-transformed before statistical analysis. Means or backtransformed means (n=4) within columns without common letters are significantly different (Tukey test, P≤0.05)

Needle N Root N Needle Ca Root Ca Needle Mg Root Mg pH in root compartment pH in hyphal compartment

Tissue concentration of mineral element (µmol (g dry weight)−1 ) or pH Nonmycorrhizal P. involutus Low N High N Low N High N

ANOVA Myc × N F value

480bc 660c 61a 40a 51a 26a 3.66a

600a 790b 29b 36a 25c 22b 3.29d

420c 880b 55a 42a 34b 29a 3.50b

590ab 1120a 35b 40a 26c 29a 3.38c

1.2 0.9 4.5∗ 0.2 18∗∗∗ 4.7∗ 60∗∗∗∗

3.90a

3.70b

3.65b

3.04c

25∗∗∗∗

Probability levels for Mycorrhiza × N supply interaction: ∗ P≤0.05; ∗∗∗ P≤0.001, ∗∗∗∗ P≤0.0001.

Foraging for N Addition of NH4 + available only to hyphae of Paxillus involutus strongly stimulated hyphal growth at the location of N application. The hyphae took up and translocated significant amounts of N to their N-deficient host plant and strongly improved host N status. Improved N nutrition, in turn, stimulated seedling growth. This series of events in our experiment provides direct evidence for the active role of the external ectomycorrhizal mycelium in foraging for N and stimulation of host N nutrition and growth. As the seedlings in our experiment were solely deficient in N, the results demonstrate that N deficiency may trigger hyphal foraging and translocation of N from N-rich sites. This is important, as, under N limiting conditions in the field, mycorrhizal hyphae would be expected to respond to gradients in soil N to effectively exploit local patches of increased N supply. Our results are in contrast to recent findings by Perez-Moreno and Read (2000). These authors have shown that Paxillus involutus associated with birch seedlings intensively colonized different sources of litter and effectively removed P rather than N from it. Although the complexity of binding forms and biochemical availability

of litter N and P complicates interpretation, these data suggest that foraging for P and not N is an important feature of Paxillus involutus. Although it is well established that fungal species and genotypes differ in their ability to respond to, and exploit, P and N sources (e.g., Bending and Read, 1995), it is likely that fungal strains have the ability to forage for more than one nutrient (George et al., 1995; Johansen et al., 1993; Wallander and Nylund, 1992). In fact, the strain employed in our work effectively responded to, and exploited, localized sources of N (this work), P (Jentschke et al., 2001), or simultaneously both nutrients (Brandes et al., 1998). The relative importance of one nutrient over the other under different conditions may be a result of host demand (Brandes, 1999). N deficiency would drive foraging for N, while in P deprived conditions the fungus may switch to searching for P. This line of arguments also applies to PerezMoreno’s and Read’s (2000) work, as their seedlings were clearly P deficient. Recent data suggest that N and P translocation in mycorrhizal fungal hyphae are interdependent within certain limits (Jentschke et al., 2001). This indicates that under realistic conditions, mycorrhizal mycelia may translocate both N and P

116 simultaneously, albeit at varying proportions, reflecting both N and P availabilty in soil patches and host demand.

oration of negative effects of high NH4 + deposition into forest ecosystems.

Mycorrhizal buffering

Acknowledgements

The seedlings were supplied with NH4 + as the sole N source. Pure NH4 + nutrition especially at elevated concentrations has repeatedly been shown to negatively influence conifer mineral nutrition and growth (Gorissen et al., 1993; Mehne-Jakobs and Gülpen, 1997; Setzer and Mohr, 1998). This has partly been attributed to rhizospheric acidification as a result of proton release during NH4 + uptake (Gill and Reisenauer, 1993). In fact, increased NH4 + supply to nonmycorrhizal seedlings increased proton concentrations in the nutrient solution, and decreased Ca and Mg concentrations in roots and needles (Table 3) in excess of that expected from dilution by increased growth. A decrease in pH in this range strongly reduced Ca and Mg concentrations in roots and needles of nonmycorrhizal Norway spruce seedlings (Godbold and Jentschke, 1998). Ectomycorrhizas may alter the cation–anion balance of coniferous seedlings (Bledsoe and Rygiewicz, 1986; Plassard et al., 1991), thereby reducing H+ extrusion during NH4 + uptake (Rygiewicz et al., 1984). As these results were obtained in solution culture experiments, where gradients in ion concentrations are generally small and hyphal ion uptake and release cannot be distiguished from root ion uptake and release, it is possible that spatial partitioning of NH4 + uptake and H+ release between root and hyphal uptake sites may contribute to mycorrhizal amelioration of NH4 + impacts. In fact, in the mycorrhizal seedlings, which acquired additional N via the mycorrhizal mycelial network, acidification in the rooting substrate was reduced compared to the nonmycorrhizal seedlings. Strong acidification in the hyphal compartment (Table 3) indicated that proton extrusion was directly linked to hyphal N uptake; thus in the ectomycorrhizal system, acidification induced by NH4 + uptake was shifted from the rhizosphere to the hyphosphere. This shift may be one of the reasons, why the additional NH4 + uptake less strongly affected cation uptake in the mycorrhizal than in the nonmycorrhizal plants. In addition, mycorrhizal hyphae may transport Ca and Mg (Jentschke et al., 2000; Melin and Nilsson, 1955) and may therefore directly contribute to improved cation uptake. Future field studies and experiments with soil grown plants may show whether the mechanisms outlined above are relevant to ameli-

We thank Christine Kettner for excellent technical assistance, and Mr. Langel and Dr. Reineking (Central Isotope Laboratory of Göttingen University) for 15 N analysis. This work was financially supported by the Ministry of Science and Culture of Lower Saxony.

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