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Plant and fungal identity determines pathogen protection of plant roots by arbuscular mycorrhizas


Journal of Ecology 2009, 97, 1274–1280

doi: 10.1111/j.1365-2745.2009.01557.x

Plant and fungal identity determines pathogen protection of plant roots by arbuscular mycorrhizas
Benjamin A. Sikes*, Karl Cottenie and John N. Klironomos?
Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada

Summary 1. A major bene?t of the mycorrhizal symbiosis is that it can protect plants from below-ground enemies, such as pathogens. Previous studies have indicated that plant identity (particularly plants that differ in root system architecture) or fungal identity (fungi from different families within the Glomeromycota) can determine the degree of protection from infection by pathogens. Here, we test the combined effects of plant and fungal identity to assess if there is a strong interaction between these two factors. 2. We paired one of two plants (Setaria glauca, a plant with a ?nely branched root system and Allium cepa, which has a simple root system) with one of six different fungal species from two families within the Glomeromycota. We assessed the degree to which plant identity, fungal identity and their interaction determined infection by Fusarium oxysporum, a common plant pathogen. 3. Our results show that the interaction between plant and fungal identity can be an important determinant of root infection by the pathogen. Infection by Fusarium was less severe in Allium (simple root system) or when Setaria (complex root system) was associated with a fungus from the family Glomeraceae. We also detected signi?cant plant growth responses to the treatments; the ?ne-rooted Setaria bene?ted more from associating with a member of the family Glomeraceae, while Allium bene?ted more from associating with a member of the family Gigasporaceae. 4. Synthesis. This study supports previous claims that plants with complex root systems are more susceptible to infection by pathogens, and that the arbuscular mycorrhizal symbiosis can reduce infection in such plants – provided that the plant is colonized by a mycorrhizal fungus that can offer protection, such as the isolates of Glomus used here. Key-words: arbuscular mycorrhizal fungi, Fusarium oxysporum, mycorrhizal function, mycorrhizal identity, pathogen protection, plant-soil interactions, root architecture

Introduction
The arbuscular mycorrhizal (AM) symbiosis is widespread among vascular plants; its bene?t to plants, however, can vary widely. Factorial combinations of different plants and fungi have experimentally veri?ed that a ‘continuum of bene?t’ occurs from parasitism to mutualism (Johnson, Graham & Smith 1997; Klironomos 2003), where bene?t is typically quanti?ed by determining the difference in growth between plants colonized with a particular fungus compared to those without the fungus. Where on this continuum, a speci?c mycorrhizal association falls is based on (i) the needs of the plant, and (ii) the ability of the fungus to perform a needed function. Placing

*Correspondence author. E-mail: bsikes@uoguelph.ca ?Current address: Biology and Physical Geography Unit, The University of British Columbia-Okanagan, Kelowa, BC V1V 1V7, Canada

a speci?c AM fungus on this continuum may be more complicated than originally anticipated. Evidence is mounting that AM fungi are multifunctional, yet we know little about the determinants of these different functions (Newsham, Fitter & Watkinson 1995b). While the main role of AM fungi in facilitating phosphorus uptake has been supported in both ?eld and glasshouse experiments (Bolan 1991; Smith & Read 1997), plants with AM fungi can also show improved water relations, reduced uptake of heavy metals and increased protection from pathogens (summarized in Newsham, Fitter & Watkinson 1995b). In some cases, these ‘alternate’ functions appear to be the primary bene?t a plant receives from the symbiosis (Fitter 1985; Newsham, Fitter & Watkinson 1995a; Singh, Adholeya & Mukerji 2000; Borowicz 2001; Herre et al. 2007). Which particular mycorrhizal function is more important may be driven by environmental factors pressuring the plant. For example, when the plant host is faced with many root pathogens but nutrients are

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Pathogen protection of plant roots by arbuscular mycorrhizas 1275
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relatively abundant, plants may bene?t more from pathogen protection. When pathogen loads are low and P is limiting (as in many glasshouse experiments), the primary bene?t of the AM association to the plant may be acquisition of P. Under these two scenarios, the same fungus would have very different functions; however, net bene?t for the plant (increased biomass or ?tness) could be similar. Recent evidence indicates that these two particular functions differ among AM fungi and correlate with their broader phylogeny (Maherali & Klironomos 2007). This result indicates that AM fungi that are best able to protect plants from pathogens would be more bene?cial under conditions of high pathogen abundance. In the absence of pathogens, these AM fungi may have a negative effect on plant growth (parasitism) due to their demand for plant photosynthate. Likewise, AM fungi that are best equipped for P acquisition may be poor partners when P concentrations are not limiting (Johnson 1993). As a result, while two different fungi could perform the same function, one fungus is more bene?cial under certain conditions. In determining a particular mycorrhizal function, both plant need and fungal ability are not mutually exclusive and are probably acting simultaneously. Thus, our goal is to test if both factors interact to determine a speci?c mycorrhizal function and if so, to what degree each determines plant bene?t. Evidence for plant-based determinants of mycorrhizal functioning is shown in the research of Newsham et al. (1995a) and Newsham, Fitter & Watkinson (1995b) who illustrated that a plant with a highly branched, ?ne root system was less dependent on mycorrhizas for nutrient acquisition. Highly branched roots should be more susceptible to infection by soil pathogens because of increased numbers of meristems and lateral roots where pathogenic fungi can invade; therefore, these plants should bene?t more from mycorrhizamediated pathogen protection. Vulpia ciliata ssp. ambigua, a plant with highly branched roots, showed reduced negative effects from both Fusarium oxysporum and Embellisia chlamydospora when inoculated with a single Glomus species (Newsham, Fitter & Watkinson 1995a). Earlier research by the same group showed that Hyacinthoides non-scripta is obligately dependent on AM fungi for its P uptake, probably due to its poorly branched root system (Merryweather & Fitter 1995). Newsham, Fitter & Watkinson (1995b) hypothesize that this poor branching would also make this species less vulnerable to infection by soil pathogens. While susceptibility to pathogens may vary among plants, their roots may be colonized by mycorrhizal fungal partners that differ in their ability to protect the plants. While many studies have now reported that plant growth bene?t depends partly on the identity of AM fungal symbionts (Sanders & Fitter 1992; van der Heijden et al. 1998; Klironomos 2003), recent evidence indicates that even the main function of the association may differ depending on the fungi involved. Both pathogen protection and P uptake can vary widely depending on the AM fungal symbiont (Garmendia, Goicoechea & Aguirreolea 2004; Vogelsang, Reynolds & Bever 2006). Maherali & Klironomos (2007) showed evidence that this variation in mycorrhizal function

is related to the broader phylogeny of the phylum Glomeromycota. In their research, AM fungi from the Family Glomeraceae were more effective than AM fungi from the Family Gigasporaceae at reducing infection by either F. oxysporum or a Pythium sp. in Plantago lanceolata. In contrast, members of the Gigasporaceae were more effective than those of the Glomeraceae at enhancing P uptake by plants. These functional differences may be a result of the distinct life-history strategies found in these two AM fungal families. The family Gigasporaceae is typi?ed by slow-colonizing species with hyphae concentrated outside the plant root, while members of the Glomeraceae colonize rapidly and usually have hyphae concentrated within the root (Hart & Reader 2002; Maherali & Klironomos 2007). While identity of AM fungi could be a determinant of mycorrhizal functioning, whether that association is bene?cial (and possibly sustained) depends on whether the plant host needs that given function. In this study, we test the hypotheses proposed by Newsham, Fitter & Watkinson (1995b) and Maherali & Klironomos (2007) – whether a single mycorrhizal function, pathogen protection, is determined by (i) the identity of plants with contrasting root architectures, (ii) the identity of the family of AM fungi with which they are associated, and (iii) their interaction. We then examine how plant bene?t differs depending on these interactions. Finally, we test one potential mechanism of pathogen protection by AM fungi. If the plant drives the function, then we predict that the coarse-rooted plant will be protected more from our pathogen than the ?ne-rooted plant, regardless of the identity of their mycorrhizal partners. Alternatively, if the fungus drives the function, then we predict that plants partnered with fungal species from the Glomeraceae will have lower pathogen levels than plants associated with species from the Gigasporaceae, regardless of plant host identity. Finally, it is also likely to be that pathogen protection is driven by the interaction between plant and fungal identity. In such a scenario, we predict that pathogen infection is reduced by a member of the Glomeraceae, but only in highly susceptible plants. For the plant growth bene?t, we predict that (i) the plant with more complex-root architecture will bene?t most from AM fungi in the Glomeraceae, because the plant has a root structure susceptible to pathogens and species from the Glomeraceae are better at pathogen protection; and (ii) a plant with a simple-root architecture will not bene?t much from pathogen-protecting species (Glomeraceae) because of its low susceptibility to F. oxysporum, but will bene?t most from members of the Gigasporaceae because of their greater potential to aid with nutrient uptake. Using our data, we were also able to test one of the proposed mechanisms for pathogen protection by AM fungi (AzconAguilar & Barea 1996). Colonization by AM fungi may compete with soil pathogenic fungi for infection sites, thus affording the plant protection (Dehne 1982; Azcon-Aguilar & Barea 1996). Increased levels of root colonization by members of the Glomeraceae could more effectively reduce pathogen infection sites (Hart & Reader 2002; Maherali & Klironomos

侵染率越高丆可有效地减少病菌的侵染位点丆从而减少病害 ? 2009 The Authors. Journal compilation ? 2009 British Ecological Society, Journal of Ecology, 97, 1274–1280

1276 B. A. Sikes, K. Cottenie & J. N. Klironomos 2007). Therefore, we predict that (i) Glomeraceae species should have greater internal root colonization than Gigasporaceae species, and (ii) after accounting for differences between plants and AM fungal families (the original treatment), the severity of F. oxysporum infection should be negatively correlated to the degree of AM fungal colonization.
PLANTS AND TREATMENT TIMING

Materials and methods
MYCORRHIZAL FUNGAL INOCULUM

Mycorrhizal spores were isolated from soils collected at the LongTerm Mycorrhizal Research Site (LTMRS) at the University of Guelph, Guelph, ON, Canada (43°32?3000 N, 80°13?0000 W). This site is an old-?eld meadow, dominated by forbs and grasses, that has been left undisturbed for more than 40 years. All six fungal isolates used in this experiment were collected from the LTMRS and maintained in glasshouse pot cultures using Allium porrum (leek) as a host. We used the following AM fungal isolates in this experiment: Glomus intraradices Schenk & Smith, Glomus etunicatum Becker & Gerdemann, Glomus clarum, Gigaspora margarita, Gigaspora gigantea, and Scutellospora pellucida (Klironomos et al. 2000).

FUSARIUM INOCULUM

尖孢镰刀菌 Fusarium oxysporum was also isolated from LTMRS soil. Soil suspension was added to Malt Extract Agar (MEA), and a variety of fungal colonies grew as a result. Several colonies of F. oxysporum were identi?ed and re-cultured on MEA. Three colonies were pooled and used in the experiment. Prior to adding F. oxysporum to the experimental units, fungal material (hyphae and spores) was inoculated onto MEA in a 1-L bottle. The fungi were left to grow for up to 6 weeks, until the colonies were covered with spores. Spores were then washed from the bottle and spore concentrations were determined using a haemocytometer.

Allium cepa (Liliaceae) and Setaria glauca (Poaceae) were used as plant hosts because they occur locally, form arbuscular mycorrhizas and have contrasting root architectures. Seeds of A. cepa were collected from plants that were introduced to a recently disturbed meadow adjacent to the LTMRS. Seeds of S. glauca were collected from a weedy roadside community next to the LTMRS. All plant seed was moistened with sterile distilled water and placed at 4 °C for 2 months prior to being introduced to the glasshouse pots. Three seeds of either A. cepa or S. glauca were germinated in each pot, and then seedlings were thinned to a single individual per pot. Plants were watered daily for the ?rst 2 weeks and subsequently watered every 2 days. After the ?rst 4 weeks, plants were fertilized weekly with 20 mL half-strength Hoagland’s solution (the full-strength solution contained (mol m)3): MgSO4, 2.0; Ca(NO3)2, 5.0; KNO3, 5.0; NH4H2PO4, 1.0, together with micronutrients and iron-EDTA) because they showed signs of nutrient de?ciency in their leaves. They were grown for 5 months to give AM fungi maximum time to establish and then inoculated with either a water control or c. 1 000 000 spores of F. oxysporum in a water suspension applied directly to plant roots using a syringe (we commonly retrieve such spore concentrations in the rhizosphere of ?eld plants from anamorphic ascomycete fungi, Fusarium spp. included). Plants were then grown for another month and harvested. After we determined wet weight, a root sample was taken for staining of fungal structures. Plants were then oven-dried at 60 °C for 2 days and weighed again to determine total plant dry weight. Dry weights were adjusted for the roots that were removed for staining.

PERCENTAGE COLONIZATION

SOIL PRE-TREATMENTS

Soils consisted of 70% sand and 30% LTMRS ?eld soil, both sterilized by autoclaving. The resulting soil mixture contained the following: NH4 = 3.8 mg kg)1; NO3 = 2.7 mg kg)1; P = 2.1 mg kg)1; K = 31 mg kg)1; pH 7.6. Soils were thoroughly homogenized and used to ?ll 1.5-L pots. To each pot, we added c. 1 g of root inoculum (chopped roots) from pot culture plants either infected with a speci?c AM fungal isolate or not infected with AM fungi as a control. Root inoculum was buried c. 1 cm below the soil surface. Each pot also received a microbial wash derived from all the pot culture soils to control for any background contaminants that are introduced with pot culture material. The microbial wash was the ?ltrate of pot culture soils suspended in de-ionized water and passed through a 20-lm sieve. Approximately 50 mL of ?ltrate was added to each pot.

Roots were stained with Chlorazol Black E (Brundrett, Piche & Peterson 1984), and percentage colonization by F. oxysporum, or AM fungi, was determined using the magni?ed intersect method (McGonigle et al. 1990). We randomly selected eighteen (2-cm long) root fragments from each pot and mounted them onto two glass slides. For each experimental unit, we assessed the presence of F. oxysporum and AM fungal structures at 150 intersections. Fusarium oxysporum was distinguished from AM fungi by the presence of linear, septate hyphae in the former compared to non-septate (or irregularly septate), knobby hyphae in the latter.

STATISTICAL ANALYSIS

EXPERIMENTAL DESIGN

Pots were arranged in a complete randomized design on a glasshouse bench. There were eight F. oxysporum–AM fungal treatment combinations (no fungal additions (control), F. oxysporum only (F only), F. oxysporum + one of the six AM fungal species (e.g. F + G. intraradices)) for each plant species (16 in total) and 10 pots per treatment combination for a total of 160 replicates.

To test for main effects of plant and fungal identity (and their interaction) on pathogen protection, we used Analysis of Variance (anova) where the percentage of root length infected by F. oxysporum was the dependent variable and plant species and AM fungal species nested within AM fungal family (Glomeraceae and Gigasporaceae) were independent factors. As AM fungal species was not a statistically signi?cant factor, we removed it from the model and re-analyzed the data. We used Tukey post hoc tests to compare F. oxysporum infection between individual plant and AM fungal family combinations. Within each plant species, we used anova and Tukey post hoc tests to compare differences in F. oxysporum infection between F. oxysporum only treatments and each F. oxysporum–AM fungal treatment. For plant biomass, we ?rst wanted to determine if infection by F. oxysporum affected plant growth. We used regression analysis to test whether F. oxysporum infection was correlated with plant biomass overall and for each plant separately (for all fungal addition treatments). For plant biomass by treatments, we used a similar anova approach as for the pathogen infection analyses to test for differences

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Pathogen protection of plant roots by arbuscular mycorrhizas 1277
between plants partnered with different AM fungal families. Within each plant species, we also tested for differences in biomass between each fungal treatment (plants without infection, infected with F. oxysporum only and each AM fungal treatment) and used Tukey tests to compare individual treatments. For differences in AM fungal colonization, we used anova to test if the percentage root length of AM fungal colonization differed between plants and AM fungal families and subsequently among F. oxysporum–AM fungal treatments with anova and Tukey post hoc tests as above. We then used regression analysis to determine if AM fungal colonization was signi?cantly correlated with the residual variation in F. oxysporum infection from our original plant and fungal identity model. We used Bonferroni corrections to account for multiple tests. Percentage colonization data for both F. oxysporum and mycorrhizal species were arcsine, square root-transformed to increase their conformance to normality. Data were analysed using the R program (R Development Core Team 2008). Graphical representations were constructed in R using the lattice plotting package (Sarkar 2008). For ?gures, percentage colonization data were not transformed.
Allium cepa Setaria glauca

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Results
Overall, we found signi?cant effects of both plant identity (P < 0.0001, F1,116 = 71.82) and fungal family identity (P < 0.0001, F1,116 = 65.63) on pathogen protection measured as infection by F. oxysporum, as well as a signi?cant interaction between these factors (P < 0.0001, F1,116 = 80.16; Fig. 1). For A. cepa, the percentage of root length infected by F. oxysporum hyphae was small when inoculated with F. oxysporum alone ( ? 15:2%). We detected no differx ence in the percentage of F. oxysporum infection between A. cepa roots inoculated with either AM fungal family (P > 0.5). In addition, there were no signi?cant differences in percentage F. oxysporum infection levels between A. cepa roots inoculated with F. oxysporum only and those inoculated with both F. oxysporum and any of the AM fungi (P > 0.5 for all pairwise comparisons; Fig. 1). In contrast to A. cepa, percentage root infection by F. oxysporum was high in roots of S. glauca inoculated only with F. oxysporum ( ? 48:7%). x Percentage root infection by F. oxysporum was equally severe in S. glauca plants inoculated with F. oxysporum and members of the Gigasporaceae ( ? 49:3%), but was signi?cantly x less when inoculated with members of the Glomeraceae ( ? 15:5%; P < 0.0001; Fig. 1). Within S. glauca, plants x inoculated only with F. oxysporum had similar infection levels to those inoculated with F. oxysporum and any member of the Gigasporaceae (P > 0.5 for all pairwise comparisons), but infection in these treatments was signi?cantly greater than in plants inoculated with any member of the Glomeraceae (P < 0.0001 for all pairwise comparisons; Fig. 1). Overall, we found a signi?cant negative correlation between F. oxysporum infection and total plant biomass (P < 0.0001, R2 = 0.299), but this relationship was strong in S. glauca (P < 0.0001, R2 = 0.570) and did not hold for A. cepa (P > 0.5, R2 < 0.001). Plant biomass was strongly in?uenced by the fungal treatments. Although we did not detect signi?cant differences

a

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Fig. 1. The effect of different fungal additions on Fusarium oxysporum infection in Allium cepa (coarse, simple roots) or Setaria glauca (?ne, branched roots). Fungal treatments are as follows: Control – no fungi added, F only – F. oxysporum only added, F + sp – effect of addition of F. oxysporum and the species indicated (Gl. intra. = Glomus intraradices, Gl. etun. = Glomus etunicatum, Gl. clar = Glomus clarum, Gi. Giga = Gigaspora gigantea, Gi marg = Gigaspora margarita, Sc pell = Scutellospora pellucida). Closed circles represent treatment median values and open circles represent 95% outliers. Boxes enclose 50% of the data between the 25th and 75th percentile, while whiskers encompass 90% of the data. Letters below the ?gure represent signi?cant differences for Tukey tests between fungal families (P < 0.001) and fungal additions (P < 0.05). [Correction added on 16 September 2009, after ?rst online publication: ?nal letter coding for AMF families corrected from ‘A’ to ‘B’].

based on plant identity (P = 0.317, F1,116 = 1.095), we did ?nd a signi?cant effect of fungal family (P < 0.0001, F1,116 = 37.31) as well as a signi?cant interaction between these factors (P < 0.0001, F1,116 = 187.69) on total plant biomass (Fig. 2). Overall, the biomass of A. cepa was signi?cantly greater when inoculated with F. oxysporum and members of the Gigasporaceae than with members of the Glomeraceae (P < 0.0001), but with some variation within fungal families. For A. cepa plants, there was no signi?cant difference in plant biomass among those individuals that were not inoculated with any fungi ( ? 1:81 g), those inoculated x only with F. oxysporum ( ? 1:92 g), and those inoculated x with both F. oxysporum and either G. intraradices ( ? 2:25 g) or G. clarum ( ? 2:17) (P > 0.05 for all x x pairwise test comparisons). Plants inoculated with both F. oxysporum and G. etunicatum ( ? 2:64) had signi?cantly x more biomass than un-inoculated plants (P < 0.05), but had similar biomass to F. oxysporum-only plants, plants partnered with other members of the Glomeraceae and those partnered with members of the Gigasporaceae (P > 0.05 for all, Fig. 2). Setaria glauca plant response was reversed, having signi?cantly greater biomass when inoculated with F. oxyspo-

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1278 B. A. Sikes, K. Cottenie & J. N. Klironomos
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Fig. 2. The effect of different fungal additions on total plant biomass of Allium cepa (coarse, simple roots) or Setaria glauca (?ne, branched roots). Biomass is not compared between plants. Fungal treatments and ?gure symbols are as in Fig. 1. [Correction added on 16 September 2009, after ?rst online publication: letter coding for ?nal two fungal addition treatments corrected from ‘b’ ‘b’ to ‘a’ ‘a’].

Fig. 3. The effect of different fungal additions on AM fungal colonization. Fungal treatments and ?gure symbols are as in Fig. 1.

zation after accounting for variation due to plant and fungal family identity (P = 0.454, R2 = 0.004).

rum and members of the Glomeraceae than plants inoculated with F. oxysporum and members of the Gigasporaceae (P < 0.0001). There was no signi?cant variation within fungal families. Biomass of S. glauca plants inoculated with F. oxysporum alone ( ? 1:46 g) was not signi?cantly differx ent from plants inoculated with both F. oxysporum and any member of the Gigasporaceae ( ? 1:61 g) (P > 0.5 for all x pairwise comparisons), whereas un-inoculated plants ( ? 3:65) and those inoculated with both F. oxysporum and x members of the Glomeraceae ( ? 3:68 g) were signi?cantly x higher (P < 0.0001 for all pairwise comparisons; Fig. 2). There was a strong interaction between plant identity and AM fungal family on the extent of AM fungal colonization (P < 0.0001, F1,116 = 31.39) as well. Although both plants had signi?cantly higher AM fungal colonization by members of the Glomeraceae (P < 0.0001, F1,116 = 213.41), in A. cepa the difference between fungal families was much greater than in S. glauca (Fig. 3). Allium cepa plants inoculated with species from the Glomeraceae ( ? 54:7%) were signi?cantly more x colonized than those inoculated with members of the Gigasporaceae ( ? 16:7%)(P < 0.0001). For S. glauca plants, x although the extent of colonization varied more by particular mycorrhizal species (Fig. 3), overall the two fungal families were still signi?cantly different (P < 0.0001, Glomeraceae   x ? 24:6%; Gigasporaceae x ? 10:1%; Fig. 3). Pairwise comparisons between individual fungal species are shown in Fig. 3. We did not ?nd a signi?cant correlation between the severity of F. oxysporum infection and the degree of AM fungal coloni-

Discussion
Our data supports the Newsham, Fitter & Watkinson (1995b) hypothesis that plant identity can determine the degree to which AM fungi can protect plant roots from pathogens. The two tested plants strongly differ in their root architecture, similar to those compared in Newsham, Fitter & Watkinson (1995b). The AM fungal partner played a larger role in protecting the root from a pathogen in the ?ne-rooted plant compared to the coarse-rooted plant. However, in addition, our data also support the hypothesis that the identity of the AM fungi in?uences the ability of the mycorrhiza to reduce pathogen infection as previously demonstrated by Maherali & Klironomos (2007). More importantly, we found that the interaction of these two factors was a major determinant of how successful a common pathogen was at infecting a plant’s root system. While our data did not explicitly address the mechanism of pathogen protection by AM fungi, we were able to test if higher levels of AM fungal colonization decreased infection by our pathogen possibly by limiting infection sites (Dehne 1982; Azcon-Aguilar & Barea 1996; Maherali & Klironomos 2007). Members of the Glomeraceae had higher percentage colonization and resulted in lower pathogen infection in our susceptible plant. However, the severity of pathogen infection in our study was better explained by the interaction of plant and fungal family identity than the degree of AM fungal colonization. In this study, we focused on a speci?c mycorrhizal function (pathogen protection). However, our data indicate that a

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F + Sc pell
c

Pathogen protection of plant roots by arbuscular mycorrhizas 1279 trade-off may exist in AM fungi among their different functions. While AM fungal-mediated pathogen protection is typically viewed as an auxiliary function, our study and others indicate that it can have strong repercussions for plant performance (Newsham, Fitter & Watkinson 1995b; Klironomos 2002; Mitchell & Power 2003). Studies suggest that negative interactions between plants and their pathogens may be a determinant of plant community structure (Klironomos 2002; Mitchell & Power 2003); however, more research is needed in this area. Thus, the ability of AM fungi to protect against such negative interactions may be equally important for plant communities. However, little is known about what edaphic factors in?uence AM-mediated pathogen protection or the relative contribution of different AM functions to plant communities. Under ?eld conditions, plants are typically colonized by multiple AM fungi at once (Daft 1983; Merryweather & Fitter 1998), but we know little about how functional complementarity of AM fungi differs between these communities (Lekberg et al. 2007; Maherali & Klironomos 2007; Jansa, Smith & Smith 2008). Our data indicate that the ability to protect plants from pathogens differs at the family level; therefore, colonization by multiple species in the same family may be redundant. However, we tested only a single pathogen and a few AM fungi, so functional variation between species (within a family) may occur for other pathogens or using more mycorrhizal species (although a larger group of AM fungi and two pathogens were tested in Maherali & Klironomos (2007) with consistent family-level divergence in pathogen protection by AM fungi). Alternatively, colonization by multiple fungal species within the same family may represent differences in colonization timing rather than functional niche complementarity. We recognize that a plant’s root architecture and its partnerships with mycorrhizas are not independent factors in nature. Indeed, nutrient limitation can induce changes in plant root morphology like increasing ?ne root hairs, but association with AM fungi can be an alternate solution (Hetrick 1991). There is evidence that colonization by AM fungi can either stimulate or inhibit root branching (Hetrick et al. 1988; Price, Roncadori & Hussey 1989; Hetrick, Wilson & Leslie 1991; Olah et al. 2005). Reduced branching is typically attributed to a decreased ability for plants to directly take up nutrients. However, it could also be a change in root morphology that is triggered by AM fungal colonization resulting in a decrease in potential infection sites for pathogens. Mycorrhizal-mediated changes in plant-root morphology for plants may be similarly based on both the degree of root plasticity for a given plant and the identity of its fungal partner. Exploring how changes in plant root architecture due to fungal colonization affect multiple AM functions may modify our understanding of below-ground feedbacks in this symbiosis. The current study was conducted using two plant species with distinct root system architecture (highly branched versus simple roots). An obvious follow-up question is whether other plant species with a wide range of root system architectures show similar responses to mycorrhizal colonization. In future studies, measures of multiple functions at the same time (e.g. pathogen protection and P uptake) could provide insight on trade-offs among different fungi. In addition, while we used only a single pathogen, multiple pathogens could be used to determine how broadly the protection occurs and to better mimic a plant’s normal soil environment. Timing of inoculations may be a key determinant of AM fungal-mediated pathogen protection particularly if priority effects determine the outcome of the interaction (Kennedy & Bruns 2005). In our study, plants were inoculated with AM fungi for 5 months prior to any pathogen addition, which ensured the AM fungi had colonized but also probably gave them an advantage. A main reason for this timing discrepancy is that we exposed the plants to AM fungi in the form of chopped mycorrhizal roots (a highly disturbed fungal mycelium), which is very different from the more intact mycelial network that plants would be exposed to in the ?eld. It is likely to be that plants are connected to an extensive and functional network very quickly in the ?eld, even with slow-growing fungi from the Gigasporaceae (Hart & Reader 2002). Nonetheless, differing the timing of AM fungi and pathogen infection may provide further insight on the mechanisms of the observed interactions. Along with a few additional taxa, Maherali & Klironomos (2007) used the same AM fungal isolates, as we did in the present study. It is interesting to note that in both studies similar responses in pathogen protection were observed, despite using different plant species (Plantago lanceolata was used in the former). However, plant biomass responses to the AM fungi were very different between the studies. This is not surprising considering the strong plant · fungal genotype interaction in plant growth response that has been observed in other studies (e.g. van der Heijden et al. 1998; Klironomos 2003). In conclusion, it is becoming increasingly clear that AM associations are multifunctional, as proposed by Newsham et al. (1995b). In this study, we show that for one function (pathogen protection), both plant identity and fungal identity can determine the outcome of the association, and that these two factors interact. Further work should focus on assessing the relative importance of different mycorrhizal functions in natural systems and the speci?c plant and fungal traits involved.

Acknowledgements
We would like to thank A. Stachowicz for technical assistance and the Natural Sciences and Engineering Research Council of Canada for ?nancial assistance. We also thank J. Powell and two anonymous referees for helpful comments on a previous version of the paper.

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