Diversity and host preference of fungi co-inhabiting Cenococcum mycorrhizae
Introduction
Ectomycorrhizae (ECM) consist of both fine root and fungal tissues, resulting in a unique and metabolically active habitat within the soil ecosystem. A healthy ECM can support a wide variety of soil organisms, including a diverse array of fungi other than the dominant ectomycorrhizal symbiont (Bergero et al., 2000, Kernaghan et al., 2003, Menkis et al., 2005, Bergemann and Garbelotto, 2006, Morris et al., 2008, Urban et al., 2008, Wright et al., 2009). These secondary fungi may be other mycorrhizal species within the root (Cázares and Trappe, 1993, Olsson et al., 2000, Morris et al., 2008, Toju et al., 2014), or saprobes on the surface and associated mycorrhizosphere (Foster and Marks, 1967, Fogel, 1988). Ectomycorrhizae also harbor a wide range of asymptomatic fungal endophytes, which colonize the root tissue internally and appear to be as common as mycorrhizal fungi (Mandyam and Jumpponen, 2005, Weishampel and Bedford, 2006, Toju et al., 2013a).
Fungal endophytes can be found colonizing all types of plant tissue (Rodriguez et al., 2009), but the species assemblages associated with roots appear distinct from those colonizing shoots and leaves (Addy et al., 2005, Summerbell, 2005). Although the ecological role of these fungi is unclear (Mandyam and Jumpponen, 2005, Mayerhofer et al., 2013), some may be latent pathogens (Schulz et al., 1999) or saprotrophs (Kernaghan, 2013). Others may provide protection from soil pathogens (Narisawa et al., 2004), or improve plant growth through phytohormone production (Khan et al., 2012), drought tolerance (Barrow, 2003), or improved access to soil phosphorus (Barrow and Osuna, 2002) or nitrogen (Upson et al., 2009).
Unlike mycorrhizal fungi, fungal root endophytes lack highly evolved absorptive structures involved in carbon and nutrient exchange (Brundrett, 2006), instead forming structures such as microsclerotia and hyphal coils within the host root (O'Dell et al., 1993, Vohník et al., 2003). Also, ECM fungal hyphae explore the soil environment and generally colonize fine roots only, while fungal root endophytes are more likely to be restricted to the roots, although they may grow throughout the root system (Menkis et al., 2004, Rodriguez et al., 2009, Toju et al., 2013a).
Much of the research into fungal root endophytes has focused on the dark septate endophytes, or DSE (Ahlich and Sieber, 1996, Grünig et al., 2002, Alberton et al., 2010, Newsham, 2011); a morphological classification encompassing fungal root endophytes with highly melanized hyphae. Although DSE are common, woody roots and ECM are colonized by a much more diverse array of fungi, including many non-melanized species, as demonstrated by several culture dependent (Schild et al., 1988, Fisher et al., 1991, Girlanda and Luppi-Mosca, 1995, Girlanda et al., 2002, Summerbell, 2005, Hoff et al., 2004, Kernaghan and Patriquin, 2011), and culture independent studies (Kernaghan et al., 2003, Kwaśna et al., 2008, Urban et al., 2008, Izumi and Finlay, 2011, Tedersoo et al., 2009, Toju et al., 2013a).
Recent evidence also indicates that some fungal root endophytes exhibit preference for particular host plants (Grünig et al., 2008, Quilliam and Jones, 2012, Kernaghan and Patriquin, 2011, Tedersoo et al., 2009, Tejesvi et al., 2013), although examples of specificity at levels seen in some ECM fungi are lacking (Molina et al., 1992, Toju et al., 2013a). There is also increasing evidence that some fungal root endophytes prefer ECM formed by particular species of ectomycorrhizal fungi (Tedersoo et al., 2009, Urban et al., 2008, Izumi and Finlay, 2011, Yamamoto et al., 2014), potentially representing a second level of root endophyte selection.
The occurrence of host preference, in combination with the range of possible effects that root associated fungi may have on their host plants, has motivated our research into differences in root associated fungal communities among host trees. In a previous study (Kernaghan and Patriquin, 2011), we investigated species composition, diversity and host preference in fungal root endophyte communities of ECM of boreal trees using culture-based techniques. To focus on differences among host plant species, and to avoid the potential influence of preference for ectomycorrhizal symbionts, ECM formed by Cenococcum geophilum (Cg) were focused on. Cg forms distinctive ECM that often dominate forest soils. It is widely dispersed, with a very broad host range and a relatively even distribution across soil horizons and successional stages (LoBuglio, 1999, Dickie et al., 2002). Although genetic variability in Cenococcum appears to be quite high (Douhan and Rizzo, 2005), the Cg ECM of a given host plant species should still represent a relatively homogeneous set of habitats for the study of root associated fungi.
Here, we revisit questions regarding the species composition, diversity and host preference of fungi associated with the Cg ECM of boreal trees, using direct PCR and cloning to eliminate the biases inherent in culture-dependant studies. We expected that the diversity of fungi associated with Cg ECM was underestimated in our previous work (Kernaghan and Patriquin, 2011) due to fast growing fungi such as Phialocephala fortinii outcompeting slower growing species. We also expected that a culture-independent approach would reveal obligate root associates that are not readily amenable to culture.
Section snippets
Sampling
ECM samples were collected from two boreal forest sites; Mount Mackenzie, Cape Breton Highlands National Park, Nova Scotia and the Lac Duparquet Teaching and Research Forest, Abitibi-Témiscamingue, Québec. The sites are 1 400 km apart and support similar mixtures of mature Abies balsamea, Betula papyrifera and Picea glauca with sparse understory vegetation dominated by herbaceous species including Cornus canadensis and Clintonia borealis, as well as ferns such as Osmundastrum cinnamomeum.
Fungi detected
Our PCR-cloning strategy detected 50 distinct non-Cg ITS sequence types that varied by at least 3 % (including 18 singletons) (Table 1, Table S1). Of these, ascomycetes accounted for 56 % of the sequence types (species) and 75 % of the amplicons and basidiomycetes represented 42 % of the sequence types and 24 % of the amplicons. The Mucoromycotina made up only 1 % of amplicons and 2 % of sequence types. At the family level, the Helotiaceae and Dermateaceae were most common, followed by the
Discussion
We detected 50 species of fungi associated with Cg ECM, and found that fungal diversity and species composition varied significantly among the three host tree species sampled. The majority of fungi detected were ascomycetes, with the Helotiaceae as the largest group overall. It is becoming clear that members of the Helotiaceae, such as Meliniomyces spp., are not only important components of fungal root endophyte communities, but are also potential ericoid or ectomycorrhizal symbionts (Vrålstad
Acknowledgments
This work was made possible by a grant from the Natural Sciences and Engineering Research Council of Canada (341671-2007). We thank Cape Breton Highlands National Park and the Lac Duparquet Teaching and Research Forest for field Logistics. We also thank Michael Mayerhofer for assistance with statistics and graphics.
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