- Top of page
- Materials and Methods
The relationship between biodiversity and plant productivity is a question that has generated much interest recently. Studies addressing the relationship between biodiversity and plant productivity have focused primarily on above-ground plant diversity (Naeem et al., 1994; Naeem et al., 1996; Tilman et al., 1996; Hooper & Vitousek, 1997; Hooper & Vitousek, 1998). However, below-ground biodiversity is also likely to play an important role in determining plant productivity. In natural ecosystems, the presence and diversity of mycorrhizal fungi can have a strong influence on plant productivity. For example, the presence of arbuscular mycorrhizal fungi altered the relationship between plant diversity and productivity in calcareous grasslands (Klironomos et al., 2000). Plant productivity has also been reported to increase at higher levels of arbuscular mycorrhizal diversity (van der Heijden et al., 1998). Although ectomycorrhizal fungi are also key components of soil ecosystems (Read, 1991; Smith & Read, 1997), the role of ectomycorrhizal diversity in determining plant productivity has only recently been investigated (Jonsson et al., 2001).
Individual tree species form ectomycorrhizal associations with a diverse community of fungal species in natural ecosystems. This diversity spans multiple scales, from the individual root to forest stands to regional and global scales (Smith & Read, 1997). Within a few centimetres on a single host root system, multiple ectomycorrhizal fungal species coexist and interact (Zak & Marx, 1964; Gibson & Deacon, 1988). In oak-pine forest stands in south-western Virginia, 138 ectomycorrhizal fungal species were identified, with 45 of these undescribed (Palmer et al., 1993). Likewise, as many as 100 different ectomycorrhizal fungal species may coexist in a single stand of Douglas fir forest (J. M. Trappe and R. Molina, unpublished results, cited in Allen et al. (1995)). The global diversity of ectomycorrhizal fungi is also relatively high, with an estimated 5000–6000 species worldwide (Molina et al., 1992).
Functionally, ectomycorrhizal fungi differ in their capacities to acquire essential nutrients from the soil and supply them to their plant hosts (Abuzinadah & Read, 1989a; Abuzinadah & Read, 1989b; Dighton et al., 1990; Dighton & Coleman, 1992; Dighton et al., 1993). For example, phosphorus uptake rates differed markedly among five ectomycorrhizal species grown in pure culture (Dighton et al., 1993) and uptake of 32P injected into distinct ectomycorrhizal root zones of birch varied according to mycorrhizal species (Dighton & Harrison, 1990). Similarly, ammonium uptake differed among Laccaria bicolor, Lactarius rufus, and Lactarius hepaticus, as demonstrated by differences in the kinetic parameters of nutrient uptake (Jongbloed et al., 1991). Ectomycorrhizal fungi also differ in their capacities to access organic forms of nutrients (Abuzinadah & Read, 1989a). High degrees of ectomycorrhizal diversity and individual differences in mycorrhizal functioning suggest that the species diversity and/or composition of these soil organisms may have important consequences for host growth and nutrition.
Few studies have examined host responses to multiple ectomycorrhizal inoculations. Chu-Chou and Grace (1985) reported that Pinus radiata seedlings inoculated with three ectomycorrhizal species had intermediate growth characteristics compared with single species inoculations. A study examining the influence of ectomycorrhizae on competition between conifer species found that the yield of competing host trees increased as the number of ectomycorrhizal fungi increased (Perry et al., 1989). Simultaneous dual inoculations of Douglas fir seedlings found that in one case seedling biomass was higher when two mycorrhizal species were present compared with one species alone (Parladé & Alvarez, 1993). Likewise, Pinus patula seedlings coinoculated with Laccaria laccata and Thelephora terrestris had higher shoot biomass compared with plants inoculated with a single fungal species (Reddy & Natarajan, 1997). A recent study by Jonsson et al. (2001) showed that under low fertility conditions growth of Betula pendula inoculated with eight ectomycorrhizal species was greater than when plants were grown with single mycorrhizal species. While these experiments suggest that the diversity of ectomycorrhizal fungi may play a role in host growth, they do not provide clear tests of the functional relationships between host responses and ectomycorrhizal diversity per se.
We investigated the influence of ectomycorrhizal diversity on plant growth and nutrient uptake by establishing a gradient of mycorrhizal diversity on experimental tree seedlings. Our approach is similar to that of Tilman et al. (1996), who established an experimental plant diversity gradient by randomly generating grassland communities of increasing plant richness. In this study, we tested the effect of ectomycorrhizal diversity on host growth and nutrient acquisition by inoculating Betula populifolia seedlings with experimental mycorrhizal ‘communities’ ranging from one to four fungal species.
- Top of page
- Materials and Methods
Using a highly simplified model system to investigate ectomycorrhizal diversity effects, the results of this study suggest that higher ectomycorrhizal diversity shifted host resource allocation to mycorrhizal root growth at the expense of shoot growth and increased P uptake in B. populifolia seedlings. Because we randomly assigned species to replicate ectomycorrhizal communities of varying diversity and included single species mycorrhizal treatments for comparison, host responses can be explained by changes in mycorrhizal richness rather than composition or sampling effect (Huston, 1997; Wardle, 1999).
Although L. laccata was dominant over other fungal species in our ectomycorrhizal assemblages, such dominance patterns are common in natural ectomycorrhizal communities (Villeneuve et al., 1989; Gardes & Bruns, 1996; Baxter et al., 1999). Indeed, we chose L. laccata in part because it was fast-growing and readily colonized plant roots. However, our choice of growth substrate, initial nutrient levels and restricted growth conditions clearly influenced fungal colonization patterns. Because initial culture conditions were the same across our diversity treatments, a similar dominant species was anticipated. Despite its relatively high levels of colonization, L. laccata did not improve B. populifolia growth over plants with other fungal species, as has been observed in other studies (Hung & Molina, 1986; Browning & Whitney, 1991), or differentially affect host nutrient acquisition. Furthermore, it showed a similar decrease in colonization at increasing mycorrhizal diversity as that of most of the other species (Fig. 3). Though L. laccata was the best colonizer and occurred more often at higher diversity levels, it did not influence host growth responses or nutrient uptake at higher fungal diversity (Fig. 5), as would be expected as a consequence of sampling effect (Huston, 1997; Wardle, 1999).
Changes in seedling growth were accompanied by an increase in overall mycorrhizal colonization but a decrease in colonization by most individual species. Reddy & Natarajan (1997) found that coinoculation increased overall mycorrhizal colonization compared with seedlings inoculated with a single mycorrhizal species. All fungi except P. bicolor showed a decrease in mycorrhizal colonization as mycorrhizal diversity increased. This indicates that mycorrhizal fungi generally colonized a smaller proportion of the root system as diversity increased. This is likely due to the lower amount of initial inoculum at higher treatment levels of mycorrhizal diversity. However, colonization by P. bicolor was not reduced by increasing ectomycorrhizal diversity, suggesting that P. bicolor was a more effective competitor than the other fungal species despite its relatively low level of colonization.
That shoot growth decreased and mycorrhizal root growth increased with greater ectomycorrhizal diversity suggests that plants allocated more carbon to mycorrhizal roots than shoots at higher levels of diversity. While increased carbon allocation to roots of mycorrhizal vs nonmycorrhizal plants has been observed (Rygiewicz & Anderson, 1994), our results suggest that carbon allocation to mycorrhizal roots might also increase at higher levels of mycorrhizal diversity. Because fungal diversity and not overall colonization best explained seedling growth patterns, greater mycorrhizal root carbon allocation may have been due to increased competitive growth of the fungi at higher mycorrhizal diversity, with a consequent increase in root and fungal respiration. In a study of the complete carbon budget of an ectomycorrhizal pine seedling, Rygiewicz & Anderson (1994) observed a shift in mycorrhizal plant carbon allocation to fine roots and fungal hyphae, and an increase in host root and fungal respiration. Yet, whether increased ectomycorrhizal diversity increases root C allocation in soils or benefits the host by increasing C production remains unclear.
Per unit mycorrhizal root biomass, root length of B. populifolia decreased as diversity increased. Because overall colonization was greater at higher fungal diversity, this could be due to hormone-induced changes in root growth and architecture (Slankis, 1973; Kottke & Oberwinkler, 1986). This is consistent with the observation that mycorrhizal roots tend to be shorter and larger diameter than nonmycorrhizal roots. Graham & Linderman (1980) reported that C. geophilum, Hebeloma crustuliniforme (Bull. Ex St. Am.), and L. laccata stimulated lateral root growth of Douglas-fir seedlings in pure culture synthesis compared to nonmycorrhizal controls, but that P. tinctorius inhibited it. Although we found no increases in root length or mycorrhizal root biomass over controls in seedlings colonized by L. laccata and C. geophilum or inhibition by P. tinctorius, seedlings colonized by P. bicolor did have higher root mass than controls.
Although mycorrhizal species characteristically differed in their colonization patterns, nonrandom variation in fungal composition resulted in few differences in B. populifolia growth or N uptake among plants colonized by different individual fungi, among distinct fungal combinations or between mycorrhizal and nonmycorrhizal plants. This suggests that the composition of ectomycorrhizal fungi on B. populifolia root systems had little effect on host growth or N uptake and that there was no significant host benefit to being mycorrhizal. This contrasts with other studies that have observed differences in host growth between mycorrhizal and nonmycorrhizal plants and among the same hosts colonized by different ectomycorrhizal fungi (Stenström et al., 1990; Browning & Whitney, 1991; Burgess et al., 1993).
The absence of a mycorrhizal benefit or effect of mycorrhizal composition on plant growth and N uptake could be due to relatively high initial N conditions in the growth medium and its subsequent depletion. High initial N availability in the medium may have lowered any potential dependency of the host on mycorrhizal colonization. However, nutrients were not added during the experiment, which resulted in lower nutrient availability over the course of the study. Based on the initial amount of N in the medium (4 mg N per Petri dish culture) and the total N taken up by B. populifolia seedlings, c. 81% of the initial available N was taken up by the plant. With decreasing N availability, competition between fungi and plants may have increased, which could explain the lack of growth benefit to mycorrhizal vs nonmycorrhizal plants. Conversely, some plants also have become more dependent upon mycorrhizal associations for access to decreasing N in the medium. One fungus, P. bicolor, appeared to enhance N uptake when it occurred on plants alone (Fig. 5d); in the four-wise diversity treatment (combination J) there was generally lower N uptake in plants without this species (Fig. 5f). Thus, P. bicolor may have acted to ameliorate potentially N limiting conditions in the growth medium.
Phosphorus uptake was higher in mycorrhizal compared to nonmycorrhizal plants and this difference increased as fungal diversity increased (Fig. 5g–i). Although B. populifolia took up a small proportion (c. 4%) of the initial P added to the medium, the capacity of the host to acquire P was enhanced for plants at higher treatment levels of mycorrhizal diversity. Phosphorus uptake also did not differ markedly among plants colonized by different mycorrhizal species or combinations of species, suggesting that mycorrhizal composition did not influence P uptake under these conditions.
Although we observed a statistically significant increase in shoot N concentration with increasing ectomycorrhizal diversity, the response was weak and variability in the data high. Of the host nutrient responses we observed, ectomycorrhizal diversity had the greatest influence on shoot P concentration in B. populifolia seedlings. Greater plant P acquisition at higher levels of ectomycorrhizal diversity suggests that the capacity of ectomycorrhizal fungi to facilitate P uptake may be enhanced at higher levels of mycorrhizal diversity. Although a mechanism for increased P uptake cannot be determined from this study, such a functional relationship between ectomycorrhizal diversity and P acquisition capacity is consistent with observed variation among ectomycorrhizal fungi in their abilities to access phosphorus (Dighton et al., 1990; Colpaert et al., 1999). To determine the potential significance of plant nutrient responses to increased ectomycorrhizal diversity, future studies should be conducted in soils under more natural conditions.
Despite somewhat greater mycorrhizal colonization at higher levels of fungal diversity, ectomycorrhizal diversity rather than overall colonization best explained variation in B. populifolia growth and nutrient uptake. This suggests that the shift in biomass allocation from shoot to root at higher treatment levels of fungal diversity was due to the diversity of ectomycorrhizal fungi rather than overall colonization. It also suggests that a greater number of ectomycorrhizal species on individual B. populifolia can enhance host access to phosphorus. While other studies have found that colonization level can affect host growth (Alexander, 1981; Newton, 1991; Timonen et al., 1997), these did not address the role of ectomycorrhizal diversity per se.
Although ectomycorrhizal colonization typically enhances plant growth (Daughtridge et al., 1986; Borchers & Perry, 1990) and nutrient uptake (Bending & Read, 1995), host responses to mycorrhizal fungi depend on a variety of factors, including soil nutrient conditions (Abuzinadah & Read, 1986) and the identity of the host and mycorrhizal symbiont (Browning & Whitney, 1991). The fact that B. populifolia seedlings were grown in axenic culture with a single initial supply of nutrients in a small volume of homogenous growth medium could have reduced the magnitude and even the direction of the mycorrhizal diversity effect. Under natural conditions, where nutrients are limiting and heterogeneous, soil volume is large, and soil organisms interact with the host and symbiont, ectomycorrhizal fungi are expected to enhance host growth and nutrient uptake. Although our study was conducted under simplified laboratory conditions, it provides the first controlled experiment examining the role of ectomycorrhizal diversity per se on host growth and nutrient uptake. If we are to extend our understanding of the role of mycorrhizal diversity to natural ecosystems, future studies must incorporate nutrient limitation, soil resource heterogeneity and soil organisms into the design and experiments conducted under natural field conditions.
While the overall benefit of ectomycorrhizal fungi to plants is well established (Smith & Read, 1997), it remains unclear to what degree and by which mechanisms ectomycorrhizal fungal diversity plays a role in governing host growth and nutrition. This study demonstrates the potential for ectomycorrhizal diversity to influence host growth and nutrient uptake in a simple axenic culture system. Given the high degree of ectomycorrhizal diversity at a variety of organizational levels and potential threats to this diversity by pollution and human disturbance (Baxter et al., 1999), a greater understanding of the functional role of ectomycorrhizal diversity is needed.