Mycorrhizal contribution to plant community composition and plant survival
We observed that AMF enhanced plant diversity and changed plant community composition by stimulating the growth of subordinate plant species (van der Heijden et al., 1998a). Eight of the 11 plant species were almost completely dependent on the presence of AMF to be successful in the microcosms. The presence of AMF stimulated their growth (van der Heijden et al., 1998a) and enhanced P acquisition (present study). However, AMF did not necessarily enhance their survival. The majority of seedlings planted at the start of the present study survived, and for those plant species with low survival (Centaurium, Trifolium, Lotus and Hieracium), at least some individuals of each plant species were still present at harvest after 20 months (Table 1). Accordingly, species richness was not affected by AMF. Instead, AMF enhanced the evenness of plant communities (Fig. 2b).
Mycorrhizal contribution to P nutrition and biomass production
We reported previously that plant productivity and P uptake in experimental macrocosms simulating North American old-field communities increased when the number of AMF taxa increased (experiment 2 of van der Heijden et al., 1998a). However, increased mycorrhizal diversity (four compared with one AMF taxa) did not result in higher biomass or in increased nutrient acquisition in this experiment. This study is in accordance with results of an earlier short-term study (3 months), where no positive effect of AMF diversity was also found (van der Heijden et al., 2003). The AMF taxa used in this study belonged to the same genus (Glomus), while the experiment with old-field plant communities (experiment 2 of van der Heijden et al., 1998a) included five different AMF genera. Variations in growth effects by different AMF taxa appear to be largest at the genus level, not at the species or isolate level (Hart & Klironomos, 2002). Moreover, it has been indicated that Glomus species have a more ruderal life style and different life-history characteristics compared with other AMF genera such as Gigaspora (de la Providencia et al., 2005). This may indicate that it is more likely to find complementary effects of AMF diversity when different AMF genera (with different strategies) are present. Future studies that test whether AMF diversity promotes plant productivity should therefore include AMF taxa from different genera.
Effects of AMF on P uptake in plant communities are poorly documented. One study showed that AMF stimulated P acquisition, and this was positively correlated with plant biomass and hyphal length density in Canadian old-field communities (experiment 2 of van der Heijden et al., 1998a). Our data show that AMF contributed to P uptake in calcareous grassland communities, and that these effects can also be independent from plant biomass. The data indicate that AMF, not only plant roots, are responsible for P acquisition: nonmycorrhizal communities contained 32% greater total root length and had a higher total biomass. Despite this, mycorrhizal communities contained 43% more P. The additional P acquisition by AMF could be explained by several factors. First, the density of mycorrhizal hyphae was 4.5–14.8 times higher than that of roots (depending on AMF treatment). Consequently, hyphae could forage for nutrients in a given soil volume much more effectively than roots. Second, hyphae can enter soil pores inaccessible to roots (Smith & Read, 1997). This might be especially important in soils rich in aggregates, as used in this experiment. Third, AMF excrete enzymes such as phosphatases and can release P from some organic sources (Joner & Johansen, 2000). Fourth, kinetic parameters (Km and Vmax) for P uptake are much higher for mycorrhizal hyphae than for roots (Jakobsen et al., 2002), and this could also explain why plant communities inoculated with AMF contained more P than nonmycorrhizal controls. Fifth, several studies performed with single plants showed that AMF interact with root system architecture (Koide, 1991). Mycorrhizal colonization can reduce root weight, specific root length and root fineness, and these effects reduce the ability of plants to acquire nutrients. Finally, AMF altered plant diversity in these grassland communities (van der Heijden et al., 1998a), and differences in P uptake could be related to changes in plant species composition between mycorrhizal and nonmycorrhizal communities (because some plant species contain more P than others). However, as AMF increased the P concentration of all plant species, this possibility is unlikely.
Plant species from different functional groups coexisted in the microcosms: several grass species with dense and fine root systems; forbs with relatively thick roots; a plant species with dauciform roots (C. flacca), and two N-fixing legumes. Despite this diversity in resource acquisition strategies, AMF appeared to be the overruling factor determining P uptake. This study thus indicates that AMF play a key role in P nutrition in grassland.
This study also shows that P nutrition depends on the identity of AMF taxa present in a community. Almost all plant species contained the highest P concentration in microcosms inoculated with AMF D, indicating that this AMF was particularly efficient in supplying P. This was also observed in a short-term pot experiment where plants inoculated with AMF D also had the highest P concentration (van der Heijden et al., 2003). In another short-term experiment with three plant species this was not observed, perhaps because a different soil was used (van der Heijden et al., 1998b). Several other studies also showed that different AMF supply different amounts of P to the plant (Jakobsen et al., 1992; Koch et al., 2004; Munkvold et al., 2004; Smith et al., 2004). AMF also altered the distribution of nutrients among co-occurring plant species, as reported earlier (van der Heijden et al., 2003). Those plants that received most P from AMF also had the highest mycorrhizal dependency (van der Heijden, 2002; data not shown). This indicates that one mechanism by which AMF stimulate growth of mycorrhiza-dependent plant species is enhanced P supply.
The increase in P uptake did not result in increased biomass production, indicating that other factors controlled plant productivity and that ‘luxury’ P consumption occurred. It has been suggested that luxury P consumption is useful for the plant because it can be utilized to enhance seed quality (Koide et al., 1988) or at other times when P is limiting (Koide, 1991). However, aboveground P storage is only successful in the absence of grazing or hay-making/mowing. The aboveground biomass of the microcosms was harvested five times at regular intervals (to simulate hay-making as in natural grassland), and substantially higher amounts of P were removed from mycorrhizal compared with nonmycorrhizal microcosms (data not shown). Moreover, the fact that enhanced P uptake did not result in increased biomass production shows that P-use efficiency was reduced when AMF were present (the average P-use efficiency of biomass was 94.3 g g−1 in microcosms with AMF and 178.6 g g−1 in nonmycorrhizal microcosms).
Mycorrhizal contribution to N nutrition
A few studies have indicated that AMF enhance plant N nutrition (Tobar et al., 1994; Mäder et al., 2000; Jin et al., 2005). It has also been suggested that AMF acquire N from organic substances by enhancing decomposition (Hodge et al., 2001). In this study we did not observe that AMF enhanced the total amount of N in plant biomass (although the experiment lasted 20 months and AMF could have acquired N from the soil, which was rich in organic matter). However, the distribution of N among co-occurring plants was influenced by AMF, and some plants received more N when mycorrhizal. One other way in which AMF could improve N nutrition of the vegetation is by stimulating growth of legumes (that fix N through their symbiosis with rhizobia bacteria; van der Heijden et al., 2006). AMF had a large effect on growth of both legume species in this experiment. However, the biomass of both species was small compared with the total biomass, and these species had a negligible effect on the total N content of microcosms. The δ15N values of both legumes were close to zero, and comparable with those of other plants (approx. 0.37), and it was not possible to calculate whether biological N fixation had occurred (despite the observation of nodules in the legume roots).
Plant productivity in terrestrial grassland is usually limited by N and/or P availability (Chapin, 1980). The N : P ratio of plant material gives an indication of which nutrient limits plant growth (Koerselman & Meuleman, 1996). An N : P ratio >14 indicates that P is limiting, while a ratio <14 indicates that N is limiting growth (Koerselman & Meuleman, 1996). The N : P ratio of the plant species in this study varied between 0.7 and 7, indicating that N availability might be the factor limiting growth in the microcosms. As expected, plants that grew in microcosms inoculated with AMF had a lower N : P ratio (because of higher P supply) compared with those that grew in nonmycorrhizal microcosms, indicating that N limitation was even greater in mycorrhizal microcosms. However, other unknown factors could also have limited biomass production in the microcosms.
Mycorrhizal contribution to soil structure
We observed that AMF reduced the loss of soil aggregates and enhanced soil aggregate stability in the microcosms. Soil structure was thus improved by the presence of AMF. This is, to our knowledge, the first experimental demonstration that AMF contribute to soil aggregation in multispecies grassland communities. Our work confirms field studies that used correlative approaches to show that AMF play a pivotal role in improving soil structure (Oades, 1993; Miller & Jastrow, 2000). Interestingly, mycorrhizal microcosms were slightly heavier compared with nonmycorrhizal microcosms (Table 2). Although not significant, this observation deserves further investigation as it may indicate that AMF reduced soil loss by preventing soil erosion during watering.
It is difficult to elucidate the precise mechanisms responsible for the effects of AMF, because many factors (plant species composition, plant nutrition, root system size, soil structure, etc.) are simultaneously affected by AMF. The effects of AMF on soil aggregation could be independent of the hyphal network, and caused by indirect effects of AMF. AMF altered plant species composition in the microcosms which, in turn, could influence soil aggregation – it is known that different plant species differ from each other in influencing aggregate formation (Degens et al., 1994).
Temporal variation in plant growth response to AMF
This study showed that growth responses of plants to different AMF were temporally variable and plant species-dependent. Lotus and Trifolium performed best with one AMF in the first growing season, but grew best with a mixture of several AMF taxa in the second. In contrast, Sanguisorba and Brachypodium responded differently to different AMF in their juvenile state (first season), while such differential effects were much weaker in the second season, when plants were older. The precise mechanisms responsible for these observations are unclear. Juvenile plants start from seed and are often nutrient-limited because they have hardly any nutrient reserves. This is in contrast to older plants that have extensive root systems and can rely on stored nutrients. Hence it is not surprising that juveniles of several plant species (e.g. Sanguisorba and Brachypodium) responded much more strongly to AMF than adult plants (Fig. 1). This observation also indicates that, in those studies where perennial plants are being studied, observed AMF effects on plant growth probably represent an upper limit because such experiments are usually performed with seedlings.
Moreover, it has been shown that particular plant–AMF combinations are most efficient in stimulating plant growth and nutrient acquisition (van der Heijden et al., 1998b; Klironomos, 2003). Such optimal plant–AMF combinations may be temporally variable and age-dependent. The fact that adult plants and seedlings of the same plant species harbour different AMF communities (Husband et al., 2002) suggests that plants have the ability to select specific AMF. Perhaps selection of specific AMF types is based on their symbiotic performance (Kiers & van der Heijden, 2006). Moreover, it may be disadvantageous for a plant to specialize on certain AMF if this benefit is temporally variable. This could explain why host specificity in the arbuscular mycorrhizal symbiosis has rarely been observed (Law, 1988; Fitter, 1990; cf. Bidartondo et al., 2002).