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There has been growing interest in the mechanistic relationship between the taxonomic diversity of ectomycorrhizal (ECM) fungi and tree productivity (Baxter & Dighton, 2001, 2005; Jonsson et al., 2001; Kipfer et al., 2012). Correlative support for a positive relationship between tree growth and ECM fungal diversity was found in our previous field study with 11-yr-old Norway spruce (Picea abies (L.) Karst), in which the root systems of fast-growing clones supported higher ECM diversity and greater root tip numbers than the roots of slow-growing clones (Korkama et al., 2006). However, in that study it was not possible to partition causality among inherent differences in host growth, differential host receptiveness and functional responses to ECM colonization, and/or differences attributable to the functional diversity of the associated ECM community.
The growth of boreal forest trees is highly dependent on their ability to gain nutrients from forest soil. ECM fungal partners of trees mobilize nutrients from soil organic matter by secreting oxidative and hydrolytic exoenzymes (Bending & Read, 1995; Read & Perez-Moreno, 2003; Rineau & Courty, 2011). These enzymes are important in the transformations of both immobile phosphorus (P) and nitrogen (N), and enable hosts to utilize not only mineral but also organic sources of N and P (Chalot & Brun, 1998). As early as 1953, Melin suggested that different ECM fungi may vary in their ability to provide nutrients to their host. ECM fungi are now known to differ greatly in their N and P uptake capabilities (Bending & Read, 1995; Baxter & Dighton, 2001; Jones et al., 2009), although some degree of functional redundancy is thought to occur within natural ECM communities (Courty et al., 2010; Pritsch & Garbaye, 2011; Rineau & Courty, 2011). In the low-nutrient, highly heterogeneous soils of boreal forests, ECM functional diversity is likely to ensure a greater uptake of nutrients.
To understand the significance of ectomycorrhizas to forest productivity, it is important to understand the underlying mechanisms operating between ECM community diversity and growth of trees. The effect of ECM species richness on host productivity (assessed as above- and belowground biomass) has been shown to be strongly context dependent (Jonsson et al., 2001; Baxter & Dighton, 2005) but potentially enhancing shoot growth (Kipfer et al., 2012) and nutrient gain (Baxter & Dighton, 2001) of seedlings. However, there are difficulties predicting the growth performance parameters of adult trees based on experimental data from seedling growth (Sonesson et al., 2002).
There is evidence that tree genotype affects the development of ectomycorrhizas and controls short-root formation of young seedlings (Marx & Bryan, 1971; Kleinschmit & Smidht, 1977; Velmala et al., 2013). Thus, the genetically controlled variation in root tip densities may imply differences in growth strategies and in the potential for growing trees to assemble ECM fungi in forest soil, which in turn could affect the efficiency of water and nutrient exchange. However, we do not know whether the variable root characteristics of spruce genotypes also include genotype-specific selection of colonizing fungi, that is, host receptivity towards fungal symbionts. In addition, it is unknown if the linkage (found by Korkama et al., 2006, 2007) between ECM community diversity and spruce genotypes differing in their long-term growth rate is apparent in seedlings before differences in their aboveground growth are visible.
Therefore, our aim in the present study was twofold. Firstly, we aimed to determine if later growth performance of spruce is related to host receptiveness to ECM fungal colonization in early stages of development. Secondly, we aimed to assess whether ECM fungal diversity, community composition, and exoenzyme capacity relate to the long-term growth rate or nutrient gain of seedlings. These questions were addressed in a glasshouse experiment using slow- and fast-growing Norway spruce seedlings.
The effect of ECM diversity on host root growth and nutrient uptake was studied by using an increasing ECM fungal diversity gradient from one to four species. In order to test the effect of genetically different host trees, two types of spruce seed orchard families were selected; three from the best performing families in long-term field trials and three from poorly performing families that have already been excluded from the official afforestation program. In addition, nutrient foraging ability determined as needle N content was used as a measure of seedling fitness.
The hypotheses were that the seedlings representing fast-growing families would: have a higher ECM colonization percentage on their roots; establish a more diverse ECM fungal community than the slow-growing ones; and that ECM taxonomic richness enhances functional diversity, and thus the ECM communities on seedlings of fast-growing families display qualitatively and quantitatively higher potential enzyme activities than those on the slow-growing seedlings.
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- Materials and Methods
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We explored the possible mechanistic role of ECM fungi in the development of Norway spruce seedlings originating from seed families known to differ in their long-term growth rate before their growth differences became apparent. The fitness of the seedlings was assessed via several approaches. First, seedling material possessing a known long-term growth performance was used. Secondly, the needle N content was determined, as this is known to function as a future resource for needle growth through positive effects on stress tolerance (Grossnickle, 2000) and on the photosynthetic activity in needles (Evans, 1989). Finally, we measured the root and shoot biomass and shoot height of the 1-yr-old seedlings at the end of the experiment.
ECM community composition of fast- and slow-growing families
During their first year of development, fast- and slow-growing spruce families did not show a distinction in receptiveness to ECM fungal richness when exposed to Wilcoxina sp., Piloderma sp., T. asterophora and T. terrestris ECM fungi. The ECM communities of the fast- and slow-growing seedlings were very similar, apart from the nursery-originating T. terrestris which showed a marginally significant trend of being slightly more abundant on the roots of slow-growing families. This is consistent with the findings of Korkama et al. (2006) in 11-yr-old fast- and slow-growing spruce clones. However, based on our results we reject our first working hypothesis and conclude that, before the differences in growth rates were visible, both the fast- and slow-growing seed families had a uniform level of ECM fungal colonization, and they established an equally diverse ECM community. In addition, we have congruent data from the same seed families inoculated with homogenized forest humus which additionally reinforce the view that in the early stages of development there is no difference in ECM diversity between fast- and slow-growing seedlings (Velmala et al., unpublished).
Effect of ECM fungi on vitality of fast- and slow-growing families
The ECM richness per se had context-dependent effects on plant production, as reported in previous experiments examining the role of diversity (Baxter & Dighton, 2001, 2005; Jonsson et al., 2001; Kipfer et al., 2012). In addition to the environmental factors already noted to affect the impact of ECM diversity on a host tree, there appears to be an internal mechanism that directs these effects, as already suggested in 1971 by Marx and Bryan. The impact of ECM richness on aboveground growth and shoot nutrient gain of fast- and slow-growing seedlings were somewhat contradictory. In the early stages of development, the slow-growing families seemed to be more responsive and to allocate the benefit from ECM richness to their aboveground parts, whereas fast-growing families allocated more of the benefit to belowground parts. Similar responses have also been observed in other studies, where poorly performing spruce families exhibited a stronger positive growth response when colonized with ECM fungi than the better performing families (Boukcim & Plassard, 2003; Mari et al., 2003). Thus, it seems that the genetic variation of spruce that is connected to later growth rates has an influence on the biomass gain that seedlings obtain from ECM fungi during their very early years. Based on these results, and in agreement with Sonesson et al. (2002), we believe that the measurements based on aboveground growth of small seedlings fail to represent the true performance of trees with a long lifespan.
Previously we reported that there is a genetic component driving the short-root formation and root growth of Norway spruce (Velmala et al., 2013). In the present study, we found that, regardless of the associated ECM community, the structure of the root systems varied between fast- and slow-growing families and was constant between growth performance groups; slow-growers had denser root systems than fast-growers. In addition, the slow-growing seedlings produced significantly more fine roots per unit root biomass and presumably less thick second- and third-order laterals, resulting in a spatially less spread-out root system. The same phenomenon was also found in our nursery study (Velmala et al., unpublished) with a larger set of seedlings from the same six spruce families.
We therefore suggest a structural effect of host genotype on the belowground ECM community via root growth and short-root structuring. It seems plausible that the associated ECM fungal communities are to a great extent determined via root structuring, rather than direct susceptibility of the host to its symbionts. Furthermore, the observed differences between the fine roots of fast- and slow-growing seedlings might later affect their ability to assemble ECM diversity in spatially heterogeneous forest soil (Peay et al., 2011). Root architecture might indeed be the ‘unknown host mechanism’ suggested by Lang et al. (2013), resulting in spatial ECM species segregation and ensuring high diversity on an individual tree. In our experimental set-up, variable root structuring was not likely to affect the colonization as the growing medium in the pots was homogenous and abundantly inoculated with ECM fungal mycelia in order to give equal opportunities for the fungi to colonize the roots.
Potential activities of exoenzymes and N acquisition
As the functional potential of ectomycorrhizas was very similar between fast- and slow-growing seedlings, one could assume that the long-term superiority of fast-growing families does not lie in utilizing the assembled ECM fungi for qualitatively or quantitatively more efficient enzyme production. Thus, our results may suggest that the belowground investment of biomass by the fast-growing families happens primarily by controlling root architecture instead of preferential selection of ECM fungi or secretion of their exoenzymes.
As expected from previous research (Bueé et al., 2007), the functional abilities of ECM short roots differed greatly according to the identity of the colonizing fungi. Hence, both the number of active short roots and the ECM fungal community composition greatly affected the total potential enzyme production of the seedlings. The species-specific fungal enzyme profiles differed especially in their potential abilities for N and P mobilization and degradation of cell wall compounds, thus indicating functional complementarity of resource use. Piloderma sp. secreted hemicellulases and produced most hydrolytic enzymes involved in the mobilization of P and N from litter and amino acids, respectively. Piloderma species have previously been reported to increase the gain of organic P and N (Baxter & Dighton, 2005). Tylospora asterophora alone was capable of high laccase activity, which has been suggested to be involved in release of N and P bound in humic substances (Criquet et al., 1999). Wilcoxina sp. showed the greatest chitinase activity and also had the highest potential activity of glucose-releasing cellulases and hemicellulases, but in turn no hydrolytic activity involved in mobilization of P and release of amino acids. Thus, from the potential enzyme activity perspective, the higher diversity treatments were superior to every single ECM fungal treatment because of an enhanced functional complementary of the more diverse ECM fungal community, a phenomenon found previously in many studies (Allen et al., 1995; Cairney, 1999; Rineau & Courty, 2011). Tylospora asterophora and Piloderma sp. were always present in our high-diversity treatments, thus adding functional abilities on top of those of T. terrestris and Wilcoxina sp., resulting in higher activities of potential P release and protein- and lignocellulose-degrading enzymes. Therefore, the higher functional activity, in the high-diversity treatments, was a matter of functional reciprocity rather than a sampling effect (Wardle, 1999).
In heterogeneous forest soils, it is advantageous for a tree to be associated with ECM fungi that can simultaneously utilize both organic and inorganic N (Kemppainen et al., 2010; Avolio et al., 2012), because ECM fungi control host gain of recalcitrant N (Talbot et al., 2013). ECM fungi increase plant productivity in nutrient-limiting conditions by enhancing access to organically bound nutrients (Jonsson et al., 2001; Baxter & Dighton, 2005) and affect both the species composition and the diversity of the root-associated fungal community through interactions with other fungi (Johnson et al., 2012). In the present study, high activity of chitinase and cellulases had a strong positive relationship with needle N content. This is consistent with the field study of Jones et al. (2009) in which the abundance of Wilcoxina sp. was associated with high accumulation of N in both shoots and roots. Based on our measurements of potential enzyme activities, Wilcoxina sp. seemed to be effective in degrading chitin, which is a structural fungal cell wall polysaccharide and an abundant organic N reservoir in boreal forest soil.
Limitations of the approach
In our study, it is possible that the dominance of Wilcoxina sp. may have masked some of the possible effects of ECM community composition and diversity on nutrient acquisition and growth in mixed inoculum treatments. This competitive reduction of ECM colonization has also been found earlier in other fungal diversity gradient studies (Baxter & Dighton, 2001, 2005; Jonsson et al., 2001). To account for this, the fungi used were selected to represent the most common ECM species found in association with young Norway spruce in natural conditions (Korkama et al., 2006, 2007) and the laborious ‘inoculation using donor ECM seedlings’ method was used. In addition, the presence of nursery-originating T. terrestris in the uninoculated treatment prevents comparisons between nonmycorrhizal and ECM-inoculated seedlings, which would be interesting but of low practical value. Nevertheless, we were still able to create a gradient of increasing ECM richness with varying fungal combinations (e.g. T. asterophora, Piloderma sp. and T. terrestris), and the resulting colonization patterns represent closely the natural diversities of small seedlings (Vaario et al., 2009). However, it is still likely that, with an even colonization of the different ECM species, the effects of each fungus contributing to the increasing ECM richness may have been clearer.
Additionally, the limitation of the enzymatic assay used is that it does not include the functional activity of the extraradial mycelia, which has a crucial role in the transfer and mobilization of nutrients from organic matter in forests compared with ECM root tips (Perez-Moreno & Read, 2000; Talbot et al., 2013). However, our comparison concerns the same fungal strains colonizing different seedlings and thus these results are comparable between seedling groups.
Our approach challenges the traditional practice of determining the performance of long-lived trees by measuring only the short-term aboveground response of seedlings. In our experiment, in which there was a relatively low richness of ECM fungi, increasing species richness increased host nutrient acquisition potential by diversifying the exoenzyme palette. However, it is likely that there is a threshold of ECM taxonomical richness over which no added value of functionality is obtained, as numerous studies suggest that some degree of functional redundancy occurs in the naturally diverse environment (Allen et al., 1995; Dahlberg, 2001; Wellniz & Poff, 2001). Our study demonstrates the importance of functional diversity of ECM species for spruce seedling nutrient acquisition. Moreover, it suggests, in accordance with the arguments of Bengtsson (1998), that neither the richness of ECM species per se nor the functional efficiency of single ectomycorrhizas is a major factor underlying the superior long-term growth of fast-growing spruce families over slow-growing ones.
It seems likely that, from a long-term perspective, faster growth rates are achieved through the additive effects of enriching factors; optimal root structuring and growth, which might lead to increased ECM diversity and more importantly ECM functional diversity, and thus better growth performance of spruce in the field. It may also be noteworthy that, during the early years of their development, the fast-growing families allocate the benefit from ECM symbiosis more to their belowground parts compared with slow-growing families. Thus, we propose that the mechanistic link between high biomass production and ECM diversity is that spruce individuals showing fast growth in the course of time possess sparse root growth and thus the potential to assemble a functionally complementary ECM community, which in a spatially heterogeneous soil results in a positive circle of accumulating benefit, also called the ‘Matthew effect’ (Merton, 1968).