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Boreal and temperate forests are generally limited by the availability of nitrogen (Tamm, 1991). Ectomycorrhizal (EM) fungi have adapted to these conditions and are efficient in their uptake and subsequent transport of N to tree roots (Smith & Read, 1997). Elevated N levels have a negative impact on the growth of many species of EM fungi; shown both in the field and in laboratory studies, and reviewed by Wallenda & Kottke (1998).
Total number and biomass of EM fruit bodies have been found to decrease in several field studies due to N fertilization of forests (Menge & Grand, 1978; Ohenoja, 1978; Wiklund et al., 1995) or the deposition of airborne N compounds (Termorshuizen & Schaffers, 1991). Some EM species (e.g. Paxillus involutus and Lactarius rufus) may on the other hand increase their production of fruit bodies in forest soils to which N has been added (Laiho, 1970; Ohenoja, 1978). EM fruit body production does, however, not necessarily reflect the activity of EM root tips and EM extramatrical mycelium (Gardes & Bruns, 1996; Jonsson et al., 2000; Dahlberg, 2001).
EM colonization of root tips tends to be influenced less by the addition of N than fruit body formation (Menge & Grand, 1978; Ritter, 1990; Brandrud, 1995). In some cases the EM colonization of root tips has been reported to be reduced shortly after N fertilization (Menge et al., 1977; Tétrault et al., 1978; Arnebrant & Söderström, 1994). However, recent studies suggest that the main effect of N addition is a shift in the EM fungal community, favoring N-tolerant species, while the frequency of root tips colonized by mycorrhizal fungi remains high following N addition (Kårén & Nylund, 1997; Jonsson et al., 2000; Taylor et al., 2000).
The external mycelium is important in increasing the surface area available for uptake and therefore a reduction in the amount of EM mycelia may reduce the uptake capacity for elements other than N (Read, 1992; Wallenda et al., 2000). The production of extramatrical mycelium by EM fungi is often reduced in response to N addition in laboratory studies (Wallander & Nylund, 1992; Arnebrant, 1994).
It is difficult to estimate the growth of EM mycelia in the field, because existing methods do not separate fungal mycelia produced by EM fungi from those produced by other groups of fungi. These problems can be avoided by the method recently described by Wallander et al. (2001), using fungal in-growth bags filled with sand. These are buried in forest soil for about 6 months or more and the nylon mesh allows fungal hyphae, but not roots, to enter. Analysis of carbon isotopes revealed that these mesh bags were colonized by mycorrhizal but not saprotrophic fungi (Wallander et al., 2001).
When nitrogen supply exceeds the requirement for the growth of forest trees, other mineral nutrients, for example phosphorus or potassium, may become limiting (Aber et al., 1989). Phosphorus deficiency results in increased allocation of carbon from shoots to roots by trees, while the opposite is true for K (Eriksson, 1995). Laboratory experiments have also shown that the production of extramatrical EM mycelia increased considerably under severe P starvation (Wallander & Nylund, 1992; Ekblad et al., 1995) but decreased under K limitation (Ekblad et al., 1995). Thus, the phosphorus status of forest trees may also influence the production of extramatrical mycelia in the field.
The objective of the present study was to investigate the influence of N fertilization on EM mycelial production under field conditions. We especially wanted to examine whether the decrease in mycelial production by EM fungi earlier found in laboratory studies after N fertilization would also be observed under field conditions. Additionally, we wanted to ascertain whether local amendment using a P containing mineral (apatite) would stimulate EM mycelial production and how this was related to N fertilization of the forest.
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Visual estimation revealed that mesh bags buried in nonfertilized plots were well colonized with mycelia after 6, 12 and 18 months with a mean degree of colonization varying from 3.0 to 3.5 (using the 0–4 classification scale) (Fig. 1a). Significantly less mycelial colonization was observed in N-treated plots at all times (mean degree of colonization between 0.0 and 0.5).
Figure 1. Fungal colonization of (a) sand and (b) sand amended with apatite (1%) in mesh bags in nonfertilized and N-treated plots based on visual estimation. Bars indicate ± SE, n = 4. Mann–Whitney U-test: N-treatment (a); P < 0.001, apatite amendment N-treated plots (a,b); P < 0.01, apatite amendment nonfertilized plots (a,b); (ns).
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Results of the phospholipid fatty acid 18 : 2ω6,9 of EM origin confirmed a significant reduction in the colonization of mesh bags in N-treated compared with nonfertilized plots (Fig. 2) (P = 0.02). The mean production of EM mycelia in N-treated plots was on average only 50% of that in the nonfertilized plots. N fertilization appeared to have a greater negative influence on EM mycelial production during 1998 than during 1999. However, the differences between the seasons were not statistically significant and the visual estimation did not reveal any interseasonal differences.
Figure 2. EM mycelial colonization of sand in mesh bags in nonfertilized and N-treated plots based on the analysis of PLFA 18 : 2ω6,9. Bars indicate ± SE, n = 4. (anova: N-treatment; P = 0.02, time; ns, N-treatment * time; ns).
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Visual estimation of mesh bags buried in root-isolated, trenched, plots revealed no colonization by fungal mycelia in N-fertilized plots (mean degree of colonisation 0.0 ± 0.0 on all harvesting occasions) and almost no colonization in nonfertilized plots (mean degree of colonization 0.5 ± 0.5 after 6 months and 0.0 ± 0.0 after 12 months). The PLFA 18 : 2ω6,9 was also low in mesh bags collected from trenched plots (0.071 ± 0.017 nmol g−1 d. wt in nonfertilised plots and 0.065 ± 0.009 nmol g−1 d. wt in N-treated plots after 6 months 1998). However, after 18 months roots and mycelium had entered some of the root-isolated plots, especially the nonfertilized plots where the PLFA 18 : 2ω6,9 contents increased to 0.281 ± 0.032 nmol g−1 d. wt (mesh bags buried in 1998) and to 0.471 ± 0.050 nmol g−1 d. wt (mesh bags buried in 1999). In N-treated plots PLFA 18 : 2ω6,9 increased 18 months after trenching to 0.220 ± 0.012 nmol g−1 d. wt (1998) and 0.217 ± 0.107 nmol g−1 d. wt (1999).
The visual estimates of fungal colonization of apatite-amended mesh bags revealed stimulated EM mycelial production by local additions of a P-containing mineral in N-fertilized plots (P < 0.01) (Fig. 1a,b). Local addition of apatite had no effect on fungal colonization in nonfertilized plots (Fig. 1a,b).
The proportion of PLFA 18 : 2ω6,9 to total PLFAs that could be attributed to saprophytic fungi in the humus samples inside the root isolated plots was 3.3 ± 0.02 in nonfertilised plots and 3.2 ± 0.24% PLFA 18 : 2ω6,9 (of total PLFAs) in N-treated plots (Fig. 3). The proportion of PLFA 18 : 2ω6,9 to total PLFAs that could be attributed to EM fungi (additional to the background) in nonfertilized plots in April 1999 was 4.1% (18 : 2ω6,9 of total PLFAs) and in October 1999 1.8% (18 : 2ω6,9 of total PLFAs) (Fig. 3). The EM biomass in humus samples in N-treated plots decreased, although not significantly (P = 0.06), to about 35% of that in nonfertilized plots (Fig. 3). Converted to biomass, EM biomass in the humus samples corresponded to c. 800 kg ha−1 in nonfertilized plots and 300 kg ha−1 in N-treated plots in April 1999, and to about 370 kg ha−1 and 120 kg ha−1, respectively, in October 1999.
Figure 3. Fungal biomass in soil samples from nonfertilised and N-treated plots expressed as the phospholipid fatty acid 18 : 2ω6,9 (% of total PLFAs). The horizontal line indicates the background due to saprotrophic fungi obtained in root-isolated plots and the biomass of EM fungi is found above the line. Bars indicate ± SE, n = 4 (April 1999), n = 8 (October 1999). (anova for EM fungi: N-treatment; P = 0.06, time; P = 0.02, N-treatment * time; ns).
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The nitrogen content of mycelia collected from in-growth mesh bags increased in N-treated plots and the C : N ratio was 20.1 ± 0.8 in nonfertilized plots and 14.8 ± 0.3 in N-treated plots (Table 2). The carbon isotopic (δ13C) value was −26.3 ± 0.4 (n = 4) in nonfertilized plots and −25.9 ± 0.0 (n = 2) in N-treated plots.
Table 2. C : N ratio of EM mycelia and rhizomorphs collected from ingrowth mesh bags in nitrogen treated (N) and nonfertilized (C) plots
| ||C : N ratio||n|
|C plots||20.1 ± 0.8||4|
|N treated plots||14.8 ± 0.3||2|
|t-test||P = 0.013|| |
The amount of ammonium extracted by 0.2 m CaCl2 was significantly higher in N-treated plots (57.2 µg g−1 organic matter, OM) than in nonfertilized plots (4.6 µg g−1 OM) when determined in April 1999 (P < 0.001) (Table 3). Nitrate concentrations were generally low, but were higher in N-treated plots (1.7 µg g−1 OM) than in nonfertilized plots (0.2 µg g−1 OM) (P < 0.001). Trenching increased the ammonium concentrations in N-treated plots (to 93.3 µg g−1 OM) (P < 0.01) and in nonfertilized plots (to 68.7 µg g−1 OM) (P < 0.001). Nitrate levels were unaffected by trenching in N-treated plots (1.5 µg g−1 OM), but increased (to 0.8 µg g−1 OM) after trenching in nonfertilized plots (P < 0.001).
Table 3. NH4+ and NO3− extracted by 0.2 m CaCl2 from soil samples collected from the organic horizon in nitrogen treated (N) and nonfertilized (C) plots in April 1999. Soil samples were collected both inside and outside root-isolated plots 12 months after trenching of roots. Means ± SE (n = 12)
| ||NH4+ (µg g−1 OM)||NO3− (µg g−1 OM)|
|C plots|| 4.6 ± 0.4||0.2 ± 0.0|
|N treated plots||57.2 ± 12.3||1.7 ± 0.2|
|t-test (treatment)||P < 0.001||P < 0.001|
|C plots, root-isolated||68.7 ± 15.1||0.8 ± 0.1|
|t-test (root isolation)||P < 0.001||P < 0.001|
| N treated plots, root-isolated||93.3 ± 10.0||1.5 ± 0.2|
|t-test (root isolation)||P < 0.01||ns|
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Almost all fungal mycelia colonizing the ingrowth mesh bags in this study were of ectomycorrhizal (EM) origin, when calculated as the difference between the fungal biomass in mesh bags collected outside, and those collected inside, the root-isolated, trenched plots. Here, only negligible amounts of fungal mycelia were produced. Furthermore, the carbon isotopic (δ13C) value in mycelia collected from mesh bags confirmed its EM origin, because the values correspond to values found in fruit bodies of ectomycorrhizal fungi in similar forests (Hobbie et al., 1999; Högberg et al., 1999; Wallander et al., 2001).
The present study clearly demonstrates that nitrogen fertilization of a spruce forest caused a significant decrease in the production of external EM mycelium. This decrease may either be an effect of reduced production of external mycelia by individual species or an effect of a changed EM community induced by the N treatment, favoring species that produce lower amounts of external mycelia. The reduction in amounts of EM mycelia produced in N-treated plots, about 50% of the production in nonfertilized plots, was of the same magnitude as that previously found in laboratory studies. In an experiment in a semihydroponics system Wallander & Nylund (1992) found a decrease in the external mycelial biomass of Suillus bovinus to 20% and of Laccaria bicolor to 35% with N in excess (100–200 mg N l−1) of that in control (1–10 mg N l−1) 8 wk after N addition started. Arnebrant (1994) found that growth of the mycelium of S. bovinus was reduced to 30% and that of Paxillus involutus to about 80% of that in control when N was added to the peat substrate at concentrations of 1–4 mg N g−1 d. wt.
In our study we found a tendency to a decrease in the amount of EM mycelia in N-treated plots according to phospholipid fatty acid analysis of soil samples. An indirect indication of reduced amounts of fungal mycelium in response to N addition at Skogaby has also been found by Lindberg et al. (2001), who noticed a reduction in the abundance of fungivorous collembolans and mites in N-treated compared with nonfertilized plots.
Interestingly, high soil ammonium levels per se did not appear to cause any decrease in EM mycelial growth, because EM mycelia and roots entered many of the root-isolated plots after 18 months of trenching (Wallander et al., 2001). This colonization occurred although the inorganic N concentration in these trenched plots was higher than the concentration found in N-treated plots (Table 3), where the growth of EM mycelia was severely inhibited. Thus, it is probably not the N concentration in soil but rather the N status of the trees that regulates growth of EM mycelia. Other factors induced by the N treatment may also have influenced growth of EM mycelia and composition of the EM community, as discussed thoroughly by Kårén & Nylund (1997). From some other studies, designed to evaluate the effect of local patches of high N on mycelial growth in forest soils, conclusions on the importance of tree nutrient status may be drawn. Stober et al. (2000) found that hyphal length and density was stimulated by local additions of N in a nitrogen-deficient forest soil, but not in a nitrogen-sufficient site. Moreover, Brandes et al. (1998) found increased EM hyphal density when nitrogen and phosphorus were added to mycelial compartments containing sand in a laboratory system with low N availability. On the other hand, Read (1991) found no increase in biomass of EM mycelium in patches with added inorganic N in forest humus in a laboratory system, probably because the humus was originally rather rich in N.
The reduction in EM mycelial production found in our study may, in part, be an effect of reduced growth of mycorrhizal fine roots. Kårén & Nylund (1997) found that the fungal biomass in EM root tips decreased, although not significantly, from 150 kg ha−1 in nonfertilized plots to 110 kg ha−1 in N-treated plots at Skogaby during 1992 and 1993. However, this was not due to a lower colonization rate by EM fungi, but to reduced biomass of fine roots following the additions of nitrogen. In any case, the more pronounced reduction in EM mycelia found in this study suggests that the influence of N on the production of external EM mycelia is probably much greater than the effect on mycorrhizal short roots (Kårén & Nylund, 1997). This has also been found in laboratory studies (Wallander & Nylund, 1992).
The influence of N fertilization on EM fruit bodies at Skogaby was very rapid and vigorous (Wiklund et al., 1995), as the production of EM sporocarps decreased from 6 kg ha−1 y−1 (mean values 1989–93) in nonfertilized plots to 0 kg ha−1 y−1 in N-treated plots. Fruit body production may be a good early indicator of the effects of N on EM fungi, but the sporocarps represent only a small fraction of the EM biomass compared with fine roots and external mycelia (Wallander et al., 2001). In conclusion, under field conditions, N fertilization affects the production of EM fruit bodies drastically; the production of EM mycelia is also severely affected, while the fungal biomass of EM root tips seems to be affected to a lesser extent.
Tree growth was initially stimulated by N fertilization at Skogaby, but after about 8 yr of nitrogen treatment tree growth in these plots started to decline compared with nonfertilized plots. At this time, tree growth was considered to be limited first by P and thereafter probably by K or Mg (Nilsson et al., 2001). Furthermore, Rosengren-Brinck & Nihlgård (1995) found an accumulation of N in old needles in N-treated plots, which they interpreted as an indication of N saturation. Our results support, to some extent, the idea that the limiting factor for tree growth has shifted from N to P, because local addition of apatite stimulated the growth of EM mycelia in N-treated plots (Fig. 1a,b). Hagerberg et al. (2003) found a similar increase in colonisation by EM mycelia of apatite-amended mesh bags in a forest with poor P status, while this was not the case in forests with a good P status.
A considerable variation in NO3 leaching from N-treated plots at Skogaby has been reported for the 2 yr of the present study; 20 mg N l−1 in the runoff during 1998 and 2 mg N l−1 during 1999 (Bergholm in: Högberg et al., 2001). This coincided with a variation in the production of EM mycelia, although time showed no significant effect in our study. The considerable loss of nitrate from the N-treated plots during 1998 could thus be the result of poor growth of the EM mycelia during this year. Although other factors may be important, the role of EM mycelium in preventing nitrate leaching should also be considered in future studies.
Needle concentrations of Mg and Ca have decreased in N-treated plots to 55–75% that in nonfertilized plots at Skogaby, and are negatively correlated to nitrate leaching (Nilsson et al., 2001). Although this decrease in Mg and Ca concentrations in N-treated plots is probably associated with nitrate leaching, the EM mycelium may also be important for the uptake of Mg and Ca, as suggested by Jentschke et al. (2000) and Blum et al. (2002).
The technique of using in-growth mesh bags enables determination of nutrient content in naturally occurring forest soil EM mycelia. We found a C : N ratio of about 20 in EM mycelia in nonfertilized plots at Skogaby, and similar C : N ratios are also reported from other forests in south Sweden (Wallander et al., 2003). The C : N ratio of EM mycelia in ingrowth mesh bags decreased to 15 in N-treated plots at Skogaby. Despite the increase in EM mycelial N content after N fertilization, the EM mycelia contained less nitrogen based on area in N-treated (3.0 kg N ha−1) than in nonfertilized plots (3.8 kg N ha−1), due to the decease in EM mycelial biomass.
In conclusion, for the first time it has been shown that N fertilization of a spruce forest has a negative influence on the mycelial growth of EM fungi in the field. This reduction was not directly related to N concentration in the soil. The N status of the trees is one possible explanation of reduced production of mycelia by EM fungi, but other factors may also be of importance. We found that local amendment with a P-containing mineral may stimulate the growth of EM mycelia in soils in N-treated forests, which have probably moved from being N-limited to P-limited as a result of the N fertilization. Our continued studies on EM mycelia in the field will include investigations of the potential of EM mycelia to retain nitrogen in forests exposed to increased input of N through deposition.