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- Materials and methods
In temperate climates, almost all species of forest trees are ectomycorrhizal. Fungal partners of these symbioses acquire fixed carbon from their photoautotrophic host, but in turn provide the host with nutrients, protect its root system from microbial pathogens, and enhance its drought tolerance (Smith & Read, 1997). Therefore, the ectomycorrhizal symbiosis is a crucial factor in forest tree health. Because this symbiosis is generally regarded as an adaptation to nutrient limited conditions (Read, 1991), the impact of air pollution in industrialized zones, particularly the enhanced deposition of atmospheric nitrogen (N), on ectomycorrhizal fungi is currently a matter of debate. A decline in species richness and abundance of macromycetous sporocarps in Europe in the 20th century has been reported, and ectomycorrhizal species seem to be particularly affected (Arnolds, 1991). Similar trends were observed in long-term inventories of macromycetes in a Swiss forest ecosystem, in which the proportion of ectomycorrhizal species decreased compared to that of the saprotrophs since the 1980s (Egli & Ayer, 1997). Enhanced N deposition was hypothesized to be the main reason for such changes (Arnolds, 1991; Rühling & Tyler, 1991). Several field experiments (overview in Wallenda & Kottke, 1998) investigating the response of the fungal community to N fertilization support this hypothesis, in that most ectomycorrhizal species had reduced sporocarp formation, whereas only minor effects were observed on saprobic species.
Using sporocarp presence as an indicator of species diversity of ectomycorrhizal fungi entails several problems (Watling, 1995): Sporocarp formation varies both in space and time and depends on a range of external factors. Detailed long-term investigations are, therefore, required to record the existing fungal community. But even if this is provided, sporocarp inventories only partly reflect the ectomycorrhizal species composition on root-systems. The lack of sporocarps does not necessarily indicate the absence of a particular fungal species at the root level (Arnolds, 1991). Furthermore, many species which do not form large, conspicuous sporocarps have been found to be important in forming ectomycorrhizas (e.g. thelephoroid, corticoid, and ascomycete fungi; Gardes & Bruns, 1996a; Kårén & Nylund, 1997; Erland et al., 1999, Jonsson et al., 1999a; Jonsson et al., 1999b; Mahmood et al., 1999).
The status of the numerous ectomycorrhizal fungi with no sporocarps above ground merit special attention. So far, species composition at the root level has been generally investigated by morphotyping the ectomycorrhizas. However, it has been shown that groupings based on macroscopic characters are often inconsistent with DNA analysis (cf. Mehmann et al., 1995; Kårén & Nylund, 1997, Jonsson et al., 1999a). The classification into morphotypes is usually too crude, and it is often not possible to draw conclusions at the species level. The PCR-RFLP analysis of the ITS region of ribosomal DNA has become a well established method for identifying fungal symbionts (e.g. Gardes et al., 1991; Henrion et al., 1992; Erland et al., 1994; Mehmann et al., 1995; Gardes & Bruns, 1996a; Kårén et al., 1997; Erland et al., 1999, Jonsson et al., 1999a; Jonsson et al., 1999b; Mahmood et al., 1999). It provides a better insight into the species composition of ectomycorrhizas and possible impacts of environmental changes on the fungal community at the root level than does morphotyping.
The question of whether the observed decrease of ectomycorrhizal sporocarps at higher N levels reflects a qualitative and quantitative reduction of these fungal taxa in the soil, that is a reduction of mycelial biomass and of the number of ectomycorrhizas formed by these species, can only be answered by investigating species diversity and abundance at the root level. There are a few studies which include belowground aspects in N-deposition investigations by monitoring morphotypes, number of mycorrhizal root tips, or ergosterol content in fine roots (Menge & Grand, 1978; Alexander & Fairley, 1983; Brandrud, 1995; Kårén & Nylund, 1997; Nilsen et al., 1998). Generally, no or only minor changes were observed belowground in response to N addition, whereas the aboveground sporocarp formation was negatively affected. In some studies, the percentage of colonized root tips was found to be reduced shortly after fertilizer application (Menge & Grand, 1978; Tétreault et al., 1978; Haug et al., 1992). In these experiments, the N fertilizer was applied at once, which aggravates the comparison to the situation of constant low inputs, for example from atmospheric deposition. To our knowledge, in only two studies has the ITS-PCR-RFLP method been applied to investigate the impact of N deposition on ectomycorrhizal fungi. Kårén & Nylund (1997) classified mycorrhizas into morphotypes, which in turn were studied using the ITS-PCR-RFLP method in a simulated N deposition experiment. Morphotype analyses indicated that a shift in species composition might occur. However, the number of RFLP-types in the examined plots did not change due to fertilization, and since they did not analyse a sufficient number of samples with the ITS-PCR-RFLP method, they were not able to draw conclusions at the level of single species. Lilleskov et al. (1998) studied the effect of enhanced N availability on above- and belowground diversity of ectomycorrhizal species over a short, steep N deposition gradient near a NH3 production facility. By ITS-typing of the fungal symbiont on root tips, they found a reduced number of species forming ectomycorrhizas at higher N levels.
In the present study, a N-addition experiment was performed using a long-term fertilizer, which should simulate a continuing, high N deposition on the forest soil. The aims of the present study were: (1) to monitor the responses of ectomycorrhizal and saprobic sporocarp production to N addition over time; (2) to determine whether the shift in the fungal species composition noted by morphotype analyses in previous N deposition studies is confirmed by ITS-PCR-RFLP-analyses and how this shift is expressed at the species level; (3) to compare the impact of N addition on above- and belowground ectomycorrhizal diversity, and in particular (iv) to see if single ectomycorrhizal species whose sporocarp production is affected by N fertilization show the same changes at the root level. The experiment was performed in a Norway spruce stand where detailed sporocarp surveys had been carried out for 3 yr before starting the fertilization.
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- Materials and methods
Sporocarp production of most ectomycorrhizal species decreased drastically after only 1 yr of N addition, whereas the saprobic community showed no distinct change. These results are in accordance with previous data on the effect of increased N input on sporocarp formation of fungi. The present study also supports the findings that the effect of N addition is much less pronounced in the belowground diversity, in that no change in the number of taxa forming ectomycorrhizas and in Simpson’s index of diversity was observed. However, a clear change of the ectomycorrhizal composition at the root level in response to increased N input was evident at the species level. Our data indicate that the reaction of single species to increased N input, as seen in sporocarp surveys, was reflected in their abundances at the root level. Even yearly fluctuations of sporocarp production were expressed in the frequencies of the ectomycorrhizas in some species. Species which produced large sporocarps in and close to the experimental plots in the 6 years of survey accounted for 23% of all sampled root tips. Such a relatively low degree of representation of the fruiting species on the root-system has also been seen in previous studies (Mehmann et al., 1995; Gardes & Bruns, 1996a; Dahlberg et al., 1997; Kårén & Nylund, 1997; Erland et al., 1999, Jonsson L. et al., 1999; Jonsson T. et al., 1999; Mahmood et al., 1999). We identified the symbiont of 44% of all root tip samples, comprising nine different RFLP-types, as belonging to the Thelephoraceae and Corticiaceae, taxa which produce inconspicuous sporocarps. Our data suggest that the proportion of these species on the root-system increases at higher N levels, whereas the proportion of species which produced fewer sporocarps after N input decreases at the root level.
Sporocarp inventories revealed a distinct change of the fungal community after only 1 yr of fertilization due to a decrease of ectomycorrhizal diversity. The sporocarp production of four of the six most dominant ectomycorrhizal species decreased drastically or even ceased. They comprise two species from the genus Russula (R. laricina, R. fuscorubroides), which has been reported to be sensitive to increased N levels in several N-deposition studies (Rühling & Tyler, 1991; Brandrud, 1995; Lilleskov & Fahey, 1996) and one species each of the genera Amanita and Inocybe (A. aff. submembranacea, I. grammata). Two of the common fruiters, Hygrophorus pustulatus and Clavulina cristata, showed no fruiting decrease after N addition. According to Arnolds (1991) and Arnolds & Jansen (1992), ectomycorrhizal fungi which mainly associate with coniferous species have shown the greatest reduction in abundance during the 20th century. These authors suggest that generalist species, forming mycorrhizas with a wide range of tree species, are less affected by increased N availability than are host-specific species. To some extent, our data confirm these findings: The two N-sensitive Russula and the Amanita species occur mainly in coniferous forests in the subalpine zone (Romagnesi, 1967; Einhellinger, 1987; Breitenbach & Kränzlin, 1991), and one of the N-insensitive species, C. cristata, is a very common species throughout Switzerland, occurring in coniferous and deciduous forests. However, I. grammata, which did reveal reduced sporocarp production, is associated with conifers as well as birch and beech (Stangl, 1989). The N-insensitive H. pustulatus presents an ambivalent situation: it is widespread throughout Switzerland, but restricted to Norway spruce stands (Breitenbach & Kränzlin, 1991), and thus is host-specific but within a broad geographical area. It might, therefore, be true that species inhabiting a more closely defined ecological niche, like the Russula and the Amanita species mentioned above, are more sensitive to N input, but a generalization is certainly not possible.
The saprobic community showed no change in response to N addition in terms of species richness and abundance. None of the species showed a clear decrease or increase of sporocarp formation in response to N addition. Rühling & Tyler (1991) noted an increase in the sporocarp production of most leaf litter and humus decomposers after N addition, particularly Clitocybe gibba and Lepista inversa. In the present study, these species occurred in the experimental area for the first time in the N-plot 2 yr after the start of N addition, which indicates that they might have benefited from higher N levels.
Mycorrhizal colonization seems to be much less sensitive to increased N input than sporocarp formation, as seen in ordination analyses. This is in accordance with other studies in which above- and belowground aspects of ectomycorrhizal fungi were taken into account (Menge & Grand, 1978; Ritter, 1990; Termorshuizen, 1993; Brandrud, 1995; Kårén & Nylund, 1997). Nevertheless, the present study confirms the findings provided by morphotype analyses that N addition has an effect on the ectomycorrhizal composition at the root level. Some RFLP-types showed significant changes in abundance 2 yr after the start of N addition. The most distinct shift can be noted for Russula laricina, which was one of the most abundant species at the root level before N supply and drastically decreased in the fertilized plots 2 yr after the start of N addition. Even more so, its sporocarp production was significantly reduced already 1 yr after starting N addition. Reduced sporocarp production seems, therefore, to be connected with or followed by decreased colonization potential at higher N levels. Laboratory and field investigations show that the infection potential of some species is decreased in response to N addition (Newton & Pigott, 1991; Arnebrant & Söderström, 1992; Arnebrant, 1996; Egli, 1996). Reduced mycelial growth is supposed to be one of the reasons for this phenomenon (Arnebrant & Söderström, 1992; Wallander & Nylund, 1992; Arnebrant, 1994).
The mechanisms by which increased N availability depresses growth processes of ectomycorrhizal fungi are not fully understood. The main hypothesis is that it affects C allocation in both partners of the symbiosis. On the one hand, C supply from the plant to the fungus can be reduced (Vogt et al., 1993) since the provision of C skeletons for N assimilation is enhanced by higher N levels in both roots and leaves (review in Champigny, 1995; Björkman, 1942). On the other hand, an additional consumption of sugars for N assimilation in the fungus itself can reduce the amount of C provided for fungal growth (Wallander, 1995). This hypothesis might also explain why saprobic species, which are not dependent on carbohydrates provided directly from living plant cells, are less affected by N addition. In the present study, N concentrations in roots and needles were significantly increased from 1.1% to 1.5% and 1.2% to 1.3%, respectively, 2 yr after the start of N addition (M. Peter, unpublished). According to the hypothesis described earlier in this section, a decrease in C supply to the ectomycorrhizal fungi caused by N addition therefore seems to be probable and might be the reason for reduced above- and belowground abundances of some species. Beside a direct impact of increased N availability, a change in the pH of the forest soil, which is usually associated with N input, may affect ectomycorrhizal fungi. In the fertilized plots, mean pH significantly decreased in the organic layer from 4.5 to 4.1 (pH in H2O; M. Peter, unpublished). Impacts of acidification are, however, thought to play a minor role in the observed decrease of ectomycorrhizal fungi in Europe (Arnolds & Jansen, 1992). Field invest-igations as well as laboratory studies show either increasing or decreasing ectomycorrhizal colonization or no clear relationship between these two variables, and it is suggested that effects of pH may differ between sites (for review see Cairney & Meharg, 1999). Since species in coniferous stands are adapted to the prevailing acidic soil conditions, it is unlikely that the relatively small decrease of pH observed in the present study was directly responsible for the decrease of ectomycorrhizal sporocarp production and root colonization of some species. A possible interaction cannot, however, be entirely excluded.
Not all ectomycorrhizal species show the same sensitivities as seen in the present study and in other field and laboratory experiments. Some species apparently can cope better with, or even profit from, increased N availability (for review see Wallenda & Kottke, 1998). In the present study, species belonging to the Thelephoraceae and Corticiaceae increased in frequency at the root level 2 yr after N addition. Similar observations, based on morphotype analyses, were obtained by Taylor & Read (1996) and Boxman et al. (1998), who noted an increase in Tylospora-like morphotypes in sites with higher N levels. Different sensitivities of species to N input might be caused by several factors. Since it is thought that a shortage of carbohydrates provided from plants for fungal growth under high N levels plays an important role, the ability to utilize other C sources might explain a better adaptation of some species. Saprophytic capabilities of ectomycorrhizal fungi have been demonstrated in several experiments (Dighton et al., 1987; Abuzinadah & Read, 1989; Durall et al., 1994; Perez-Moreno & Read, 2000). For example, Tylospora fibrillosa was shown to have decomposing and proteolytic abilities (Ryan & Alexander, 1992; Cairney & Burke, 1994). Sporocarps of resupinate ectomycorrhiza formers, which include most members of the Thelephoraceae and Corticiaceae, are commonly developed on litter and soil debris and on well-decayed wood, perhaps suggesting saprophytic abilities (Erland & Taylor, 1999).
We do not know if the observed reduction of ectomycorrhizal frequencies of some species in response to N addition will persist and if species might even be out-competed by better adapted ones. Since previous long-term studies about the impact of increased N levels on belowground species composition have been conducted by studying morphotypes only, information at the species level is scarce. Experiments in which existing N levels were decreased by sod-cutting (Baar, 1996) or by means of a roof and application of clean throughfall water (Boxman et al., 1998) have shown that species richness measured by sporocarp formation can be restored. This indicates that species might persist at the roots in unfavourable environments. Lilleskov et al. (1998), however, found a reduced number of ectomycorrhizal species at the roots of trees growing in a forest close to a NH3 production facility and receiving high inputs of N for 28 yr. On the long run, it might therefore be possible that some species will disappear at continuing N addition. In the present study, the N input was quite high (150 kg N ha−1 yr−1). The question arises whether the observed decrease of sporocarp formation of ectomycorrhizal fungi in lower N-input studies (Brandrud, 1995; Boxman et al., 1998) is also reflected at the roots at the level of species. Morphotype analyses indicated no shift in the belowground species composition in these low N-level experiments with inputs of 30–40 kg N ha−1 yr−1 (Brandrud, 1995; Brandrud & Timmermann, 1998, Wallenda & Kottke, 1998), but it is possible that changes were overlooked by this method. The functional significance of a change in species composition at the fungus–root interface and probably also in the abundance of mycelia in the soil for the trees and the whole forest ecosystem is not known. A deeper insight into the physiological properties of different ectomycorrhizal species, which result in unequal sensitivities to the available amount of inorganic N, is necessary and could possibly provide answers to this field. In the present study, belowground ectomycorrhizal diversity was not reduced by N addition, but a shift in abundance at the root level from species forming large sporocarps to species which produce no or resupinate sporocarps took place and might get more pronounced at a continuing input. Even if ectomycorrhizal functions for the trees are still provided with these species, an irreversible loss of fungal species may occur in the long run. Considering this, present N-input levels up to a mean of 35 kg N ha−1 yr−1 as found in Swiss forests (Flückiger & Braun, 1998) might therefore be problematic.
We noted a good correlation between changes in above- and belowground abundances for some species. Even yearly fluctuations of sporocarp production were reflected in the belowground frequencies. The same correlation was observed in previous studies (Laiho, 1970; Agerer, 1990). This supports the hypothesis of Dahlberg et al. (1997) who assumed that a part of the among-year variation of sporocarp production not accounted for by annual variation in temperature and precipitation (Dahlberg, 1991), may be due to fluctuations in the mycorrhizas. Some species (Amanita aff. sumbembranacea, Inocybe grammata, Russula fuscorubroides, Clavulina cristata), however, were common in terms of sporocarp production but were either never found on roots or, in case of R. fuscorubroides, identified only in two root samples. For Clavulina cristata, the reason for the mismatch might be intraspecific variation in the ITS region. The unknown RFLP-type 4 of the present study clustered with the cantharelloid types from the ML5/ML6 database of Bruns et al. (1998). This group is closely related to Clavulina cristata (Burns et al., 1998). In fact, the location of the root tips on which we detected this RFLP-type corresponded exactely to the observed patches of Clavulina sporocarps. Furthermore, ITS-RFLP patterns were identical in one restriction enzyme and similar in the other two. For the other species, a possible explanation for the mismatch between above- and belowground abundances could be that they might be mainly present on roots at lower depth than the sampled layer (approx. upper 5 cm). Egli (1981) studied the vertical distribution of ectomycorrhizal morphotypes and noticed some types to be restricted to specific vertical soil layers. In relation to the study reported here, we took some soil cores and sampled mycorrhizas which occurred at lower depth in the organic layer (between approx. 5–10 cm) and showed an unfamiliar morphotype. Among these samples, we were able to identify I. grammata, which had not been found in the previously. No RFLP pattern matched the one of A. aff. submembranacea, however, so this species might occur at even lower depth. Gardes & Bruns (1996a), who also found a mismatch between above- and belowground frequencies of some species, suggested that the discrepancy might result from different patterns of resource allocation among species. The efficiency in acquiring carbon from the host plant may vary among ectomycorrhizal species or additional access of saprobic sources of carbohydrates may result in different proportions of mycorrhizas for the same amount of sporocarps. If additional saprobic capabilities were the reason for the observed mismatch between above- and belowground abundances, one would assume that these species might be less affected by increased N input, which, however, was not the case in the present study. Insights into vertical stratification of species, together with information about the effect of N on single species, might therefore provide better knowledge of nutrient and C allocation of different species.
Ectomycorrhizal species richness is markedly higher below- than aboveground in the present study, where 68 different RFLP-types compared with 25 fruiting species were found. Of the sampled ectomycorrhizas > 75% were formed by species which did not produce conspicuous sporocarps in the investigated plots. This is in accordance with previous studies (Mehmann et al., 1995; Gardes & Bruns, 1996a; Dahlberg et al., 1997; Kårén & Nylund, 1997, Jonsson et al., 1999a; Jonsson et al., 1999b; Mahmood et al., 1999; Taylor & Bruns, 1999). In all these investigations, mycorrhizas formed by species of the Corticiaceae and Thelephoraceae accounted for a large proportion of ectomycorrhizal root tips. In the present study, these resupinate taxa as well as the Russulaceae were dominant on root systems in untreated plots, which seems to be a general pattern of EM communities in California (Taylor & Bruns, 1999). Our data indicate that N addition has an influence on the ratio of these two groups of species in favour of the resupinate taxa. The functional significance of these until recently overlooked resupinate species thus deserves even more attention in the light of their role in N-polluted regions.