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Below-ground plant pathogens, parasites, herbivores and mutualists influence the performance and competitive ability of plant species and their offspring (Klironomos, 2002; Reinhart et al., 2003; De Deyn et al., 2004). Plants also selectively affect the soil biota associated with their rhizosphere (Wardle, 2002) and, as a consequence, feedback interactions are established between plants and soil organisms. These interactions are crucial for the spatial and temporal composition of natural plant communities (Gange & Brown, 2002; Bever, 2003). The sign of these feedbacks (positive or negative) can also change depending on the multitrophic interactions between different rhizosphere organisms (van der Putten, 2003). However, below-ground interactions that include more than one group of soil organisms have been largely ignored in ecological studies.
In coastal sand dunes, soil-borne pathogenic fungi and root-feeding nematodes are responsible for the degeneration of Ammophila arenaria, which in turn leads to plant succession (van der Putten et al., 1988, 1993). Coastal foredunes are highly dynamic ecosystems characterized by severe wind-driven sand accretion. Not only is A. arenaria resistant to sand burial, but it needs sand accretion in order to maintain vigorous growth. Sand burial allows A. arenaria to avoid ageing by developing new roots (Marshall, 1965) and provides the opportunity to escape temporarily from root-pathogens and herbivores (van der Putten et al., 1990). Although root-feeding nematodes gradually colonize the new sand layers, there is a lag of 4–5 wk in which the new roots grown in a freshly deposited layer of wind-blown beach sand are in an ‘enemy-free’ environment (de Rooij-van der Goes et al., 1998; van der Stoel et al., 2002). When sand accretion stops, as in stabilized dunes, root pathogens and herbivores accumulate in the rhizosphere leading to a gradual degeneration and eventual disappearance of A. arenaria.
Among the root-feeding nematodes that accumulate in the rhizosphere of A. arenaria, the endoparasitic genus Pratylenchus (root lesion nematode) is of special relevance. This genus has a wide distribution along the Atlantic and Mediterranean coasts in Europe (Karssen et al., 2001), and occurs at relatively high densities in later stages of dune succession (Zoon et al., 1993). Pratylenchus are migratory endoparasitic nematodes that invade, multiply, feed and move on the root cortex of the host plant resulting in necrotic lesions and promoting fungal infections (Back et al., 2002). Moreover, Pratylenchus penetrans, which is considered a serious pest on a wide range of economically important crops (Pinochet et al., 1996), is also supposed to be a key factor in the die-out of the North-American Ammophila breviligulata (Seliskar & Huettel, 1993).
The deleterious effect of root-feeding nematodes on plant growth is dependent on the combination of nematode species (Brinkman et al., 2005), as well as on the density of nematodes in the rhizosphere. However, in dune soils the density of root-feeding nematodes is considerably lower than that observed when nematodes are added to plants growing in sterilized soil (de Rooij-van der Goes, 1995; Brinkman et al., 2004). Such nematode control in natural systems may be explained by bottom-up mechanisms (by the host-plant), top-down control (by natural enemies) and control by plant mutualists (e.g. arbuscular mycorrhizal fungi and endophytes).
The role of arbuscular mycorrhizal fungi (AMF) as protective agents against root-feeding nematodes has been tested in crop plant species with highly variable results (Hol & Cook, 2005). One major limitation of those studies is the use of commercial strains of AMF which had not coevolved with the crop and the nematodes. The diversity of AMF found in natural communities might be important for the outcome of the interaction because of the functional differences between different AMF taxa (Klironomos, 2003). In coastal sand dunes, AMF account for 30% of the total soil microbial biomass (Olsson & Wilhelmsson, 2000). It is therefore reasonable to assume that they play an important role in these systems. Based on studies with A. breviligulata and Leymus arenarius, arbuscular mycorrhizal fungi are considered to be a major candidate for nematode control in foredunes (Little & Maun, 1996; Greipsson & El-Mayas, 2002), but no data are available for most other sand dune plant species or for the putative mechanisms involved in nematode control.
Plant protection by AMF might be caused by physical and physiological plant responses to the fungal infection (Graham, 2001). Alternatively, AMF could have a direct suppressive effect on root-feeding nematodes if both organisms compete for root space and feeding sites (Francl, 1993). We used A. arenaria because it is one of the very few wild plant species for which there is a wide knowledge about interactions with root-feeding nematodes. Two experiments were designed to study the mechanisms by which AMF may control root-feeding nematodes, using A. arenaria and P. penetrans as model organisms. In the first experiment we examined whether preinoculation with AMF makes plants more tolerant to herbivory or provides an increase in plant resistance to the herbivores. In the second experiment, we analysed the importance of the presence of arbuscular mycorrhizal fungi and P. penetrans in the same root compartment of A. arenaria for the outcome of the interaction. The results obtained from both experiments provide new clues to understand the mechanisms of control of root-feeding nematodes by AMF in natural systems.
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Our results show that native AMF can protect A. arenaria through the suppression of P. penetrans colonization and reproduction. The process of P. penetrans suppression by AMF acts through locally operating mechanisms. In other studies on coastal dune systems, Greipsson & El-Mayas (2002) found that a commercial AMF inoculum protected the dune grass Leymus arenarius against migratory endoparasitic nematodes. Little & Maun (1996) showed that mycorrhizal protection of Ammophila brevigulata against Pratylenchus and Heterodera spp. was effective when sand burial occurred simultaneously. The majority of studies on the interaction between AMF and Pratylenchus spp. have been done with perennial crops and the results are inconsistent. Some showed increases in plant tolerance or resistance to Pratylenchus spp. as a consequence of plant inoculation with AMF, but others did not find any protective effect of AMF (Roncadori, 1997; Forge et al., 2001; Elsen et al., 2003).
The data presented here show, for the first time, that AMF can outcompete migratory endoparasitic nematodes when they occur together in the same root compartment; this contrasts with previous studies with migratory endoparasitic nematodes in which AMF appeared to enhance nematode multiplication (Borowicz, 2001). Conversely, root colonization by AMF was not affected by the migratory endoparasitic nematodes, so we did not detect mutual inhibition between AMF and nematodes as proposed previously (Francl, 1993). The detailed mechanisms of suppression of nematodes were not analysed; however, our results suggest that direct competition with AMF hyphae in the root or local changes in root chemistry or exudates may have been responsible for the inhibition of nematode reproduction (Graham, 2001).
Some authors have hypothesized that AMF protection is only effective if plants are colonized by the mycorrhizal fungi before the attack by pathogens and/or herbivores. This hypothesis is based on the improved nutritional and health status of mycorrhizal plants which allow them to support higher densities of root-feeding nematodes (Azcón-Aguilar & Barea, 1996; Vaast et al., 1998). We did not find a higher concentration of N and C in the plants that were preinoculated with AMF 2 wk and 5 wk before nematode inoculation, but plant biomass was significantly higher in the FN5 treatment than when nematodes and AMF were inoculated simultaneously. However, this positive effect of AMF preinoculation might have occurred through nematode suppression and not through increased plant tolerance because the effect of preinoculation with AMF was a further reduction in nematode reproduction and infection.
Increases in plant growth through improved plant nutrient uptake are considered to be the main benefits that plants obtain from the symbiosis with AMF (Jeffries et al., 2003). A significant increase in plant growth was observed in the first experiment but not in the second one. This disparity might be caused by differences in the AMF species between both experiments, but also, and more likely, by the different age of the plants used in the two experiments, 2 wk vs. 8 wk, because younger A. arenaria plants display a greater response to AMF (Rodríguez-Echeverría et al., 2004). Thus, changes on plant biomass and nutrient content between treatments were not as severe in the split-root experiment as in the first experiment. The biomass allocated below ground was 20% in the sequential inoculation experiment and 40% in the split-root experiment. The proportion of biomass allocated above and below ground by a plant species depends on environmental factors, plant age and growing time (Klepper, 1991). Because pot size was different in both experiments, the variation in the percentage of below-ground biomass could be explained not only by plant age but also by the greater sand volume that each plant had in the split-root experiment. It is noteworthy that in both experiments the proportion of biomass allocated below-ground increased with the inoculation of nematodes and/or AMF.
The presence of nematodes did not have a negative impact on plant growth. However, nematodes wiped out the beneficial effect of AMF and affected plant N and C content. These differences were again greater in the sequential inoculation experiment, probably because young plants are more sensitive to the attack by root-feeding herbivores (van der Putten et al., 1990). Plants in the sequential inoculation experiment also reallocated N and C above-ground when attacked by the nematodes, a common reaction in plants subjected to important root damage (Masters & Brown, 1997). In the split-root experiment the highest N content was found in plants infected only by nematodes. Although these plants were probably more tolerant to herbivory than the 2-wk-old seedlings, this increase in N content can be considered an indicator of plant stress (Whittaker, 2003), as observed for A. arenaria when growing in nonsterilized soil (van der Putten et al., 1988). Root colonization by AMF did not increase nutrient content in the plants of the split-root experiment, a fact that could be explained by the lower responsiveness of older seedlings combined with the lower values of root colonization by AMF.
The AMF communities associated with the roots of A. arenaria were very similar in both experiments, containing mainly Glomus sp. from the Glomus-group A (Schüßler et al., 2001). The genus Glomus comprises the majority of species within the phylum Glomeromycota. Glomus species are also more resistant to disturbances than other genera of AMF (Dodd et al., 2000). Therefore, the AMF communities detected on the roots probably represent the fraction of field inoculum that can survive and grow in our experimental conditions. As in other molecular studies of AMF colonizing plant roots, our sequences did not correspond to previously described AMF species suggesting a higher natural AMF diversity than acknowledged from culture collections. Our understanding of the importance of AMF diversity for the symbiosis is still limited, however, the interactions of plants with complex natural AMF communities are probably richer than with commercial AMF inocula. Studies addressing ecological issues should not underestimate the importance of the natural high diversity of AMF.
The ability of AMF to control P. penetrans in the rhizosphere of A. arenaria could be crucial under natural field conditions. A study by van der Stoel et al. (2002) showed that root-feeding nematodes, including Pratylenchus spp., accumulate in 4–5 wk after the growth of new roots in the new fresh sand layer. They found that the density of nematodes (and other adverse soil organisms), after a month of the sand deposition, could significantly reduce growth of test plants in glasshouse trials. However, they also observed that in the field, mature A. arenaria can overcome that negative effect of nematodes in the foredunes. Arbuscular mycorrhizal fungi were excluded from their glasshouse trials, but our results demonstrate that the interaction between A. arenaria and root-feeding nematodes cannot be fully understood without AMF.
The diversity of organisms involved in below-ground interactions makes it difficult to single-out the direct implications and effects of different groups; however, our results show that AMF can control root herbivores associated with the grass A. arenaria. This mechanism can be added to the bottom-up and interspecific competition processes that have been previously reported as regulatory of nematode populations in coastal dunes (Brinkman et al., 2004). The role of nematode antagonists, the effect of AMF in other nematode genera and the consequences of this interaction for nematode competition needs further consideration to completely understand nematode control in natural systems.