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Testing the link between community structure and function for ectomycorrhizal fungi involved in a global tripartite symbiosis Jennifer K. M. Walker1,2, Hannah Cohen1, Logan M. Higgins1 and Peter G. Kennedy1,3 1
Department of Biology, Lewis and Clark College, 0615 SW Palatine Hill Rd, Portland, OR 97219, USA; 2Hawkesbury Institute for the Environment, University of Western Sydney, Locked
Bag 1797, Penrith, NSW 2751, Australia; 3Department of Plant Biology and Ecology, Evolution, and Behavior, University of Minnesota, St Paul, MN 55108, USA
Summary Author for correspondence: Peter G. Kennedy Tel: +1 612 624 8519 Email:
[email protected] Received: 29 September 2013 Accepted: 10 November 2013
New Phytologist (2014) 202: 287–296 doi: 10.1111/nph.12638
Key words: Alnus rubra, community structure and function, ectomycorrhizal fungi, Frankia bacteria, Pseudotsuga menziesii, root tip exoenzyme assays, tripartite symbiosis.
Alnus trees associate with ectomycorrhizal (ECM) fungi and nitrogen-fixing Frankia bacteria and, although their ECM fungal communities are uncommonly host specific and species poor, it is unclear whether the functioning of Alnus ECM fungal symbionts differs from that of other ECM hosts. We used exoenzyme root tip assays and molecular identification to test whether ECM fungi on Alnus rubra differed in their ability to access organic phosphorus (P) and nitrogen (N) when compared with ECM fungi on the non-Frankia host Pseudotsuga menziesii. At the community level, potential acid phosphatase (AP) activity of ECM fungal root tips from A. rubra was significantly higher than that from P. menziesii, whereas potential leucine aminopeptidase (LA) activity was significantly lower for A. rubra root tips at one of the two sites. At the individual species level, there was no clear relationship between ECM fungal relative root tip abundance and relative AP or LA enzyme activities on either host. Our results are consistent with the hypothesis that ECM fungal communities associated with Alnus trees have enhanced organic P acquisition abilities relative to non-Frankia ECM hosts. This shift, in combination with the chemical conditions present in Alnus forest soils, may drive the atypical structure of Alnus ECM fungal communities.
Introduction There has been considerable progress in recent years documenting the spatial and temporal patterns of ectomycorrhizal (ECM) fungal communities, a dominant microbial symbiosis of plants in most temperate and many tropical forests (Peay et al., 2008). Attempts to understand the processes driving these patterns have followed, with a range of abiotic and biotic factors being recognized as significantly influencing the richness and composition of ECM fungal communities (Koide et al., 2011; Tedersoo et al., 2013). Although the factors driving ECM fungal community variation are becoming better understood, the link between the structure and function of ECM fungal communities remains less well resolved (Koide et al., 2007; Courty et al., 2010a). Connecting these two variables is a major goal in ecological studies of both micro- and macroorganisms (Robinson et al., 2010; Thompson et al., 2012) and will be essential in predicting how both plants and their microbial symbionts respond to increasing environmental change (Deslippe et al., 2011; Pickles et al., 2012). The assessment of the link between the structure and functioning of ECM fungal communities has been slowed by the inability to grow a wide range of ECM fungal species in laboratory settings (Courty et al., 2010a). In addition, many of the assays quantifying ECM fungal functioning are not designed for large-scale analyses, making community-level inferences challenging (Pritsch Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
& Garbaye, 2011). Recently, a high-throughput method that measures potential exoenzyme production on excised ECM fungal root tips, followed by molecular identification of the fungal species present, has helped address both of these issues (Pritsch et al., 2004, 2011; Courty et al., 2005). The enzyme assay allows researchers to connect both individual- and community-level ECM fungal identity with enzymatic function in relation to a number of organic nutrient sources, including carbon, nitrogen (N) and phosphorus (P) (Pritsch & Garbaye, 2011). Since its initial development, the assay has been used to examine ECM fungal functional responses to a range of abiotic and biotic variables (Courty et al., 2010a, 2011; Jones et al., 2010; Herzog et al., 2012). Unlike many other ECM hosts, the genus Alnus is involved in symbioses with both ECM fungi and N-fixing Frankia bacteria. The structure of ECM fungal communities associated with Alnus trees is exceptional, in that they are both species poor and highly host specific. This unusual pattern has been well demonstrated at local, regional and global scales (Tedersoo et al., 2009; Kennedy et al., 2011; P~olme et al., 2013; Roy et al., 2013) and, although differences in ECM fungal communities between Alnus and hosts in the Pinaceae have been shown repeatedly (Miller et al., 1992; Tedersoo et al., 2009; Kennedy et al., 2011), differences have also been demonstrated recently between Alnus and Betula ECM fungal communities (Bent et al., 2011; Bogar & Kennedy, 2013). New Phytologist (2014) 202: 287–296 287 www.newphytologist.com
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These latter results suggest that the atypical structure of Alnus ECM fungal communities is not an artifact of host phylogenetic similarity (Tedersoo et al., 2013). Multiple mechanisms may be responsible for the observed pattern, but differences in ECM fungal functioning as a result of differences in the nature of each microbial symbiosis (i.e. dual vs tripartite) could be an important selective factor differentiating Alnus and non-Alnus ECM fungal communities. Given the potential overlap of ECM fungi and Frankia bacteria with respect to host N acquisition, multiple researchers have suggested that ECM fungal communities associated with Alnus trees are especially proficient at P acquisition, which may co-limit N fixation and plant growth (Chatarpaul et al., 1989; Molina et al., 1994; Horton et al., 2013). Some support for the enhanced P acquisition hypothesis was shown by Yamanaka et al. (2003), who found that Alnus seedlings colonized by both Frankia and the ECM fungal species Alpova diplophloeus could have higher P tissue concentrations when grown in certain types of soil than seedlings colonized by Frankia alone. However, as the presence of ECM fungi can increase seedling P levels regardless of host (Smith & Read, 2008), it remains possible that the response seen in the study of Yamanaka et al. (2003) was caused only by ECM fungal colonization, and not specifically because of an ECM fungus specialized for greater P acquisition. In addition to potential effects on ECM fungal P acquisition, it is well established that the presence of Frankia bacteria significantly changes both the abiotic and biotic environment experienced by ECM fungi. Alnus forest soils are typically enriched in inorganic N (Miller et al., 1992; Compton et al., 2003, Mitchell & Ruess, 2009), which is more readily assimilated by ECM fungi relative to organic N (Smith & Read, 2008). In addition, significant quantities of Frankia-derived N have been detected in the hyphae of ECM fungi present on the same Alnus host plant (Arnebrant et al., 1993; Ekblad & Huss-Danell, 1995). Both of these results suggest that Alnus-associated ECM fungi may be less reliant on organic N acquisition from soils relative to ECM fungi associated with non-Frankia hosts, and therefore lower their N-related enzyme production. The aforementioned features of the Alnus tripartite symbiosis make it an attractive system to explore the link between ECM fungal community structure and function. To determine whether Alnus-associated ECM fungi have a differential ability to acquire organic nutrients, however, comparisons with ECM fungi on non-Frankia ECM hosts are needed. In the forests of the Pacific Northwest, USA, Alnus rubra Bong. (Red alder) is a dominant tree species in early successional settings and is typically followed by the non-Frankia ECM host Pseudotsuga menziesii (Mirb.) Franco (Douglas fir; Miller et al., 1992). Previous studies of these two hosts have indicated that their ECM fungal communities differ in both richness and composition, and that co-colonization by the same ECM fungal species is rare (Miller et al., 1992; Horton et al., 2005; Kennedy & Hill, 2010). Although the aforementioned enzyme assay has been used to assess the response of P. menziesii ECM fungi to fertilization (Jones et al., 2010), to our knowledge, it has not yet been used to explore how ECM fungal community function differs on hosts with dual vs tripartite microbial symbioses. New Phytologist (2014) 202: 287–296 www.newphytologist.com
In this study, we compared the potential enzyme activity of field-collected ECM fungal root tips sampled from A. rubra and P. menziesii in adjacent monodominant stands. Our aims were to test whether ECM fungal communities associated with A. rubra differed in their ability to access organic P and N when compared with ECM fungi on P. menziesii and to assess how the relative abundance of individual ECM fungi correlated with the relative exoenzymatic activity. We hypothesized that, because of the copresence of Frankia bacteria, the A. rubra ECM fungal communities would have increased P and decreased N acquisition abilities relative to the ECM fungal communities present on P. menziesii. In addition, we hypothesized that, because of a benefit to the host plant of enhanced nutrient acquisition by ECM fungi, the most abundant ECM fungi on both hosts would show higher P and N acquisition abilities than those that were less abundant.
Materials and Methods Site description The experimental sites were established between 1984 and 1987 in the Coast Range (Cascade Head) and Cascade Mountains (H.J. Andrews) of western Oregon, USA. Cascade Head is located < 2 km from the Pacific Ocean at 330 m elevation; the mean annual precipitation (MAP) is 270 mm and mean monthly temperature (MMT) ranges from 5°C to 16°C. H.J. Andrews is located further inland in the Cascade mountain foothills at a higher elevation (730 m) and in a somewhat drier (MAP = 260 mm) and more variable (MMT = 1–18°C; Knowe & Hibbs, 1996) climate. The andisol soils at Cascade Head are derived from weathered basalt, and composed of loam and interspersed duff up to 1 m in depth, whereas the inceptisol soils at H.J. Andrews are formed from igneous rock and volcanic ash, and characterized as gravelly loam over cobbly silt loam (Radosevich et al., 2006). Before establishing the experimental sites, the second growth forest at each site was clear-cut, burned and replanted with a series of 27 9 27 m2 plots of various mixtures of A. rubra and P. menziesii, including replicated monodominant plots of each host (Knowe & Hibbs, 1996). Sample collection and root tip selection We sampled only the monodominant plots of both ECM host species at the two experimental sites in spring (April/May) and summer (June/July), 2012. At both sites, fine roots were collected from soil samples within 1 m of the base of eight trees at two replicate plots for each tree species (2 seasons 9 2 sites 9 2 tree species 9 2 replicate plots 9 8 soil samples per plot 9 10 root tips per sample, n = 1280). Trees were sampled along transects that were 10 m from each other and from the plot edge, and trees chosen along the transect were always at least 5 m apart. In one A. rubra plot at H.J. Andrews, which had only a few solitary trees because of poor establishment, some soil samples were taken < 5 m from P. menziesii, but roots were confirmed to be from A. rubra by the presence of Frankia nodules. Soil samples ranged from 15 cm 9 15 cm 9 20 cm deep to 30 cm 9 30 cm 9 5 cm Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
New Phytologist deep, but always resulted in the removal of approximately the same volume of soil. Each soil sample (hereafter referred to as a core) was removed by hand, placed in a plastic bag and kept intact at 4°C until prepared for enzymatic activity assays, which occurred within 72 h of sampling. On the same day that enzyme assays were performed, roots were gently rinsed under cold tap water and cut into 1-cm segments. Root segments from each core were placed in a gridded tap water-filled dish and randomly sampled until nine live ECM fungal root tips and one non-ECM fungal root tip were collected per soil core (ECM and non-ECM tips were distinguished by standard macromorphological traits, such as the presence/absence of root hairs, root swelling, mantle formation and the presence of emanating hyphae). Clean, turgid ECM fungal root tips were cut to 2–4 mm using tweezers under a dissecting microscope. As soon as the root tip enzyme assays were complete, DNA was extracted from every root tip for subsequent molecular identification (see later). Root tip enzyme assays Enzyme assays were based on the methods developed by Pritsch et al. (2004), expanded by Courty et al. (2005) and improved by Pritsch et al. (2011). All root tips were assayed for the potential activity of acid phosphatase (AP) and leucine aminopeptidase (LA). Solutions, substrates and standards were prepared according to published protocols and kept at 20°C until use. The rinsing and incubation buffers for the 4-methylumbelliferonebased enzyme substrate (4-methylumbelliferyl phosphate) was set at pH 4.5, and for the 7-amino-4-methylcoumarin-based enzyme substrate (L-leucine-7-amino-4-methylcoumarin hydrochloride) was set at pH 6.5, for maximum efficiency. The stopping buffer used was 1.0 M Tris base dissolved in sterile deionized water; 96well filter plates (Pall Corp., Port Washington, NY, USA) containing root tips, controls for background fluorescence and wells for the calculation of a standard curve were subjected to rinsing, incubation and measurement. The incubation time was 15 min for AP and 1 h for LA, and took place at 21°C in darkness. Fluorescence measurements were carried out using an LS 55 Fluorescence Spectrometer fitted with a Plate Reader accessory and FL Winlab software (Perkin-Elmer, Waltham, MA, USA) at an excitation wavelength of 364 nm, an emission wavelength of 450 nm, slit widths of 5 nm, at 230 V. The 1% attenuator setting was used to decrease the sensitivity of the spectrometer 20-fold based on internal optimization. Following enzyme assays, the root tip surface area was determined with WinRhizo software (Regent Instruments Inc., Sainte-Foy, QC, Canada) and the rate of potential enzyme activity was calculated in pmol mm 2 min 1. Molecular analysis and sequence processing Total genomic DNA was individually extracted from each assayed root tip using the REDExtract-N-Amp Plant kit (SigmaAldrich, St Louis, MO, USA), and the internal transcribed spacer (ITS) region of nuclear rDNA was amplified using the fungalspecific primer pair ITS1F and ITS4. Results were stained with Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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SYBRSafe (Life Technologies, Grand Island, NY, USA) and visualized on 1.5% agarose gels. If more than one band appeared on the gel, the basidiomycete-specific primer pair ITS1F and ITS4B was used in a second PCR. For faint bands, the initial DNA was diluted by a ratio of 1 : 10, and a second PCR was performed. Samples with single bands of the expected length were cleaned using 0.5 ll of ExoSAP IT (USB Corp., Cleveland, OH, USA). Cleaned PCR product was then submitted for Sanger sequencing at the University of Arizona, USA Genetics Core sequencing facility (UAGC) using the forward primer ITS1F. Sequences were manually edited and trimmed by visually inspecting each chromatogram; they were subsequently assembled into contigs using a 97% similarity threshold in Sequencher Version 5.1 (Gene Codes Corporation, Ann Arbor, MI, USA). Contig representatives and singletons (i.e. operational taxonomic units (OTUs)) were submitted to the INSD and UNITE fungal DNA databases for identification. The OTUs were named to species if they had a 97% or greater match to database sequences over at least 450 bp; discretion based on the length of the match and source of the database sequence was used to identify sample sequences with matches between 93% and 96% (often named to genus) and between 90% and 92% (mostly named to order). The identities and accession numbers of the best database match for the representative sequences of the ECM fungal species detected are provided in Supporting Information Table S1. All tips that were not identified because of a lack of sequence data were eliminated from the final analysis: this resulted in the loss of a few replicate tips in several cores per plot and, rarely, the loss of an entire core. In total, only nine cores had fewer than three replicate tips and five plots had up to two cores lost. A subsample of tips was also assessed to confirm plant host identity using the trnL chloroplast region, following the methods of Kennedy et al. (2011); nine non-target host tips were eliminated from the analysis, all of which were from A. rubra plots at H.J. Andrews. Soil nutrient status Soil samples were collected from all plots at both sites for chemical analyses in spring (April) 2013. Five 500-g replicate samples were collected along transects at each plot, as described for the core sampling above (i.e. transects were at least 10 m from the plot edge and from each other, and samples were at least 5 m from each other). Fresh samples were sent to the Oregon State University Central Analytical Laboratory for analysis of pH, inorganic (Bray) and total (Kjeldahl) P, available ammonium (NH4) and nitrate (NO3), and total N. Data analysis The sampling design involved four factors: host (A. rubra, P. menziesii), site (Cascade Head, H.J. Andrews), season (spring, summer) and plot (two replicates per host). Because individual ECM fungi typically have patchy distributions (Taylor, 2002), we chose to sample two plots at each site to try to capture as broad a range of the ECM fungal community present on both hosts as possible. We were not formally interested in assessing New Phytologist (2014) 202: 287–296 www.newphytologist.com
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spatial variation among plots within a host, or in characterizing differences in overall community composition, but rather focused on determining whether there were differences caused by host, site and season, and their interactions. In preliminary analyses of ECM fungal community composition, we found that statistical models were functionally identical for the three variables of interest whether or not plot was included (Tables S2, S3). This indicated that the variation between plots was not a biologically meaningful factor for our analyses, and so we averaged over root tips within a core and cores within a plot for the analyses of ECM fungal community composition and potential enzyme activity. Statistical models included fully factorial combinations of season, site and host, with all factors considered to be fixed. Variation in ECM fungal community composition (based on presence–absence data) was tested using a hierarchical multivariate permutational ANOVA (Anderson, 2005). Differences in community composition between sites and hosts were visualized with Nonmetric Multidimensional Scaling (NMDS) in R version 2.15 (R Core Development Team, Vienna, Austria). Variation in ECM fungal community potential enzyme activity for LA and AP was assessed with separate three-way ANOVAs in Statistica 6.1 (StatSoft Inc., Tulsa, OK, USA). To meet normality assumptions, the raw ECM fungal activity data of both enzymes were cube root transformed before analysis based on Shapiro–Wilks’ tests. For both the composition and activity analyses, post-hoc Tukey’s honestly significant difference (HSD) tests were used to assign significant differences among factor means. A number of ECM fungal species were encountered with sufficient replication (i.e. present in n ≥ 3 cores per site per host) to examine the relationship between the relative abundance and relative potential enzyme activity of ECM fungal communities. For this analysis, each host was analyzed separately, using five species on A. rubra and 15 species on P. menziesii. Relative abundance and relative potential enzyme activities were calculated for each of these species by dividing individual species values by the mean species abundance or the mean raw enzyme values of the selected species pool on each host. With this metric, relative abundance and activities above and below one represent greater or lesser relative community contributions, respectively (i.e. compared with a theoretical average ECM fungal species in each host community). The potential enzyme activity of individual ECM fungal species was also used to calculate the proportional contribution to community enzyme activity of each species on each host when abundance and enzymatic activity were considered together (as in Courty et al., 2010b). This value was derived by dividing the activity of each species per host by the total community potential activity (i.e. the sum of activity for all dominant species per host). The proportional contribution to ECM fungal community potential enzyme activity for each species was then calculated by multiplying this value by the relative abundance of each species (i.e. the number of soil cores in which each species was detected compared with the total number of soil cores per host). To assess variation in the soil chemical properties between sites and hosts, we tested each variable with a fixed-factor two-way ANOVA (R version 2.15), with significant differences between means New Phytologist (2014) 202: 287–296 www.newphytologist.com
determined with post-hoc Tukey’s HSD tests. In all analyses, results were considered to be significant at P ≤ 0.05.
Results ECM fungal community structure From the 1280 root tips collected, 992 yielded a PCR product of sufficient quality for sequencing and 888 were identified. Of these, 790 were colonized by ECM fungi, with identification success being 62% for A. rubra and 75% for P. menziesii. A total of 98 OTUs were identified as ECM fungal species, with 30 species present in at least 5% of cores sampled per host. The dominant ECM fungal species on A. rubra included Alnicola sp. 1, Tomentella spp. and Lactarius obscuratus, and on P. menziesii included Cenococcum geophilum and Russula sp. 1 (Table S4). There were 18 species present exclusively on A. rubra, 76 exclusively on P. menziesii and four were shared. Phylogenetic analysis of the dominant shared taxon, Lactarius obscuratus, showed that root tip samples clustered distinctly by host species (Fig. S1), suggesting that the 97% sequence similarity threshold was not sufficient for clear taxonomic designation in this case. The three other shared taxa (Cortinarius sp. 2, Tomentella sp. 1 and Tomentella sp. 3) were present on only a single tip of one of the two hosts. Given these limitations, no conclusions about the enzymatic performance of an ECM fungal species when colonizing different hosts could be inferred. With regard to ECM fungal community composition, there was a significant interaction between site and host (Table S3). Although the ECM fungal communities differed significantly between the two hosts at both sites (pairwise post-hoc tests; P ≤ 0.02), an NMDS plot revealed that the A. rubra ECM fungal communities were more similar between Cascade Head and H.J. Andrews than those on P. menziesii (Fig. S2). ECM fungal root tip potential enzyme activity At the community level, the potential AP activity of ECM fungal root tips on A. rubra was significantly higher than that of those on P. menziesii at both sites (Fig. 1a; Table S5). Potential AP activity was also significantly higher at Cascade Head than at H.J. Andrews in the spring, but not significantly different between the sites in the summer (Table S5; Fig. S3). Potential LA activity differed between sites for each host, and was significantly higher on P. menziesii ECM fungal root tips at Cascade Head, but not significantly different between hosts at H.J. Andrews (Fig. 1b). Season had no significant effect on potential LA activities (Table S5; Fig. S3). Individual ECM fungal species varied considerably in potential enzymatic activities (Fig. 2). Most species on both hosts had similar responses for both enzymes (i.e. consistently lower or higher than the community mean); however, some differed notably in their relative potential AP and LA activities. Specifically, Clavulina sp. and Piloderma sp. 2 had approximately twice as much relative potential AP activity than potential LA activity, whereas Alpova diplophloeus, Helvella lacunosa, Hygrophorus sp. and Lactarius rubrilacteus demonstrated the reverse pattern. The Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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relative potential enzyme activities that were equivalent or above the community mean for at least one of the enzymes assayed. Although the ranking of this relationship stayed relatively similar for the A. rubra ECM fungal communities when root tip abundance and enzyme activity were multiplied, the contribution of rarer ECM species on P. menziesii to community enzyme activity dropped considerably when both were considered together.
Acid phosphatase activity (pmol mm–2 min–1)
(a) 300
250 a 200
a
150
b
b
Soil nutrient status Soil P, N and pH differed significantly between sites and between hosts (Table 1). Soil collected from A. rubra plots and from Cascade Head had consistently higher inorganic P and N, as well as lower pH, than that collected from P. menziesii plots and from H.J. Andrews, although total P and nitrate were not significantly different (Table 1b).
100
50
0
Discussion Leucine aminopeptidase activity (pmol mm–2 min–1)
(b)
80
Community-level symbiont interactions
60 a
40
b
ab
ab
20
0
Cascade Head
H.J. Andrews
Fig. 1 Potential acid phosphatase (a) and leucine aminopeptidase (b) activities for the ectomycorrhizal fungal communities on Alnus rubra (closed boxes) and Pseudotsuga menziesii (open boxes) at Cascade Head and H.J. Andrews (western Oregon, USA). Raw data are presented in the figure, but all data were cube root transformed to meet assumptions of normality in statistical analyses. Lower case letters designate significant differences at P ≤ 0.05 based on Tukey’s honestly significant difference (HSD) tests. Boxes surrounding median values represent the first and third quartiles, and whiskers show the smaller (and larger) of either the maximum (and minimum) values or 1.5 9 the interquartile range (c. 2 SD).
relationship between root tip abundance and enzyme activity varied somewhat between hosts, particularly for the less abundant species. On A. rubra, the dominant taxon, Alnicola sp. 1, and the rarer species, Cortinarius sp. 2 and Alpova diplophoeus, had relative potential AP and LA activities that were both lower than the community mean. By contrast, on P. menziesii, the dominant taxon, Clavulina sp., and multiple rarer species, including Rhizopogon vinicolor, R. villlosus, R. rogersii and Tuber sp., had Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
We found that the ECM fungi associated with A. rubra and P. menziesii differed in terms of community structure, as well as organic P and N acquisition. At both of the study sites, the ECM fungal communities on A. rubra had higher P potential enzyme activities and, at Cascade Head, lower N activities relative to those on P. menziesii. The consistency of the P-related results, in spite of significant differences in both community composition and abiotic environmental conditions across sites, suggests that these two host species have preferential association with ECM fungal communities differing in P acquisition ability. The N-related results reinforce the notion that the ECM fungal communities on A. rubra and P. menziesii can differ significantly in potential enzyme activity. Although these ECM hosts differ in many aspects of their ecology, aside from the nature of their microbial symbioses, the nutrient acquisition patterns found here support the hypothesis that the co-presence of Frankia bacteria may influence the functioning of Alnus-associated ECM fungal communities. Specifically, the higher P acquisition ability of Alnus-associated ECM fungi may be the result of a greater host demand for this nutrient, given the steady N supply provided by the Frankia bacteria. Similarly, the more muted N acquisition ability of the A. rubra ECM fungal communities at Cascade Head is consistent with a lowered host and fungal demand, resulting from the co-presence of an N-fixing symbiont. Complementarity between ECM fungal and N-fixing actinorhizal microbial symbioses has been shown by Chatarpaul et al. (1989), who found that inoculation of Alnus incana seedlings with Frankia bacteria and the ECM fungus Paxillus involutus together resulted in significantly better total growth than individual inoculations. Comparable results have also been found consistently for legume hosts simultaneously colonized by N-fixing rhizobial bacteria and arbuscular mycorrhizal (AM) fungi (Kaschuk et al., 2010; Larimer et al., 2010). In addition to effects on host plant growth, complementary nutrient contributions may also influence individual symbiont performance. Rose & New Phytologist (2014) 202: 287–296 www.newphytologist.com
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2.0 1.5 1.0
0.04
Alpova 0.02 diplophloeus
0.03
Cortinarius sp.2 0.03
0.15
Lactarius 0.18 obscuratus
0.19
Tomentella 0.25 ellisii
Tomentella 0.34 testaceogilva
0.23
0.0
0.36
0.5 Alnicola sp.1 0.17
Relative to community mean
(a) 2.5
Relative to community mean
0.01 0.02
0.01 0.02
0.02 0.03
0.02 0.04
0.01 0.04
0.01 0.03
0.03 0.03
0.04 0.03
0.10 0.10
0.14 0.07
0.01 0.08
0.08 0.05
0.06 0.10
0.18 0.20
2.5
0.28 0.17
(b) 3.0
2.0
1.5
1.0
Youngberg (1981) found that mycorrhizal colonization was needed for optimal Frankia performance; in particular, P uptake by AM fungi was correlated with higher N fixation. Similarly, Diagne et al. (2013) showed that colonization by ECM fungi resulted in greater numbers of Braydrhizobium nodules and higher leaf N content on experimentally inoculated Acacia mangiam seedlings. Conversely, Koo et al. (1995) demonstrated that Frankia nodulation was critical for ECM fungal colonization of A. rubra, and both Arnebrant et al. (1993) and Ekblad & Huss-Danell (1995) found that Frankia-derived N was present in the amino acids in the hyphae of the co-colonizing ECM fungus Paxillus involutus. Although these studies collectively indicate that mycorrhizal fungi and Frankia bacteria do interact, if only New Phytologist (2014) 202: 287–296 www.newphytologist.com
Rhizopogon rogersii
Rhizopogon villosulus
Tuber sp.
Rhizopogon vinicolor
Helvella lacunosa
Hygrophorus sp.
Rhizopogon parksii
Clavulina cristata
Lactarius obscuratus
Piloderma sp.2
Lactarius rubrilacteus
Russula sp.1
Cenococcum geophilum
Leucangium carthusianum
0.0
Clavulina sp.
0.5 Fig. 2 Relative root tip abundance (black bars) and potential acid phosphatase (gray bars) and leucine aminopeptidase (white bars) activities of ectomycorrhizal fungal species present in > 3 cores per site per host on Alnus rubra (a) and Pseudotsuga menziesii (b). A value of 1 represents the community mean (solid line). Numbers in or above each column represent the proportional contribution to community enzyme activity. See the Materials and Methods section for details on how each metric was calculated.
indirectly, exactly how resources are traded and which partner drives the trading dynamics has yet to be determined. As the host contributes carbon to both of these partners, the P- and Nacquiring attributes of each symbiont may be driven by host plant selection (Kiers et al., 2011), but further experiments in which symbiont interactions are structured as direct (e.g. colonizing the same root space on a shared host) or indirect (e.g. colonizing different root space and only interacting through a shared host) would be helpful in providing additional insight into the specific nature of these tripartite interactions. Differences in the nature of the symbioses between hosts appeared to be an important factor driving differences in potential enzyme activities, but a range of other factors probably Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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Table 1 Differences in soil phosphorus (P), nitrogen (N) and pH between Alnus rubra and Pseudotsuga menziesii plots at Cascade Head and H.J. Andrews where hosts and/or sites differ (a) and where host and site interact (b) (a) Site or host1
pH2
Inorganic P (ppm)
Ammonium N (ppm)
Total N (%)
Cascade Head H.J. Andrews P-value A. rubra P. menziesii P-value
4.5 0.1 5.4 0.1 < 0.001 4.7 0.1 5.2 0.1 < 0.001
22.8 3.6 17.7 2.3 0.501 24.0 3.2 16.6 2.7 0.065
21.6 3.7 11.2 1.3 0.015 20.3 3.6 12.5 1.9 0.025
0.81 0.06 0.56 0.08 0.001 0.89 0.06 0.49 0.06 < 0.001
(b) Site 9 host1 Cascade Head A. rubra P. menziesii H.J. Andrews A. rubra P. menziesii P-value
Total P3 (ppm)
Nitrate N (ppm)
1766 206a 2482 290a
34.0 4.6a 16.6 3.3a
1732 100a 1135 145b < 0.001
29.2 6.2a 1.6 0.5b < 0.001
1 Soil samples were collected from two replicate plots per host per site (n = 5, N = 2). 2 Raw values SE are presented for all variables, but those measured in ppm were log transformed before ANOVAs. 3 Lower case letters denote a significant difference at P ≤ 0.05 derived from post-hoc Tukey’s honestly significant difference (HSD) tests of the interaction terms.
contributed to the differences observed between sites and seasons. We speculate that the higher total soil N at Cascade Head was the primary driver of the significantly higher potential LA activity of P. menziesii ECM fungal communities relative to those on A. rubra. As already noted, the greater reliance of P. menziesii on ECM fungi for organic N acquisition (because of the absence of an N-fixing symbiont) would probably favor an ECM fungal community with increased responsiveness to shifts in soil N availability. Changes in ECM fungal community composition between sites may also have affected the N acquisition patterns observed on P. menziesii; however, the lack of significant differences in community potential enzyme activity between Cascade Head and H.J. Andrews suggests that this effect was relatively minor. Enzyme activity in temperate forest soils is known to be limited by both temperature (Brzostek & Finzi, 2011; Baldrian et al., 2013) and moisture (Brockett et al., 2012), and the later onset of warmer wetter weather at H.J. Andrews could explain the lower potential AP and LA enzymatic activities on both hosts in the spring at that site. In addition to temperature and moisture effects, the variation in enzyme activities observed by season may also be a response to temporal fluctuation in host and/or fungal nutrient demand. For example, in a Quercus-dominated forest in France, Courty et al. (2007) showed that ECM fungal enzymatic activities were temporally variable and related to host growth patterns. Similarly, in a mixed hardwood forest in the USA, ECM fungal community enzyme activity responded to a seasonal increase in soil nutrients (Burke et al., 2011). Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
Individual-level symbiont interactions Studies of ECM fungal functioning have long noted that enzyme activities can vary considerably among individual species, but the relationship between ECM fungal species abundance and potential enzymatic activity is less well understood. As it might be expected that ECM hosts would prefer to associate with ECM fungal species with higher enzymatic activity (to maximize host nutrient acquisition), it was somewhat surprising to observe that Alnicola sp. 1, the most abundant ECM fungal species on A. rubra, had relatively low potential AP and LA activity. The enzymatic performance of Clavulina sp. on P. menziesii was somewhat better, particularly for potential AP activity, but it also did not have the highest relative root tip activity in the P. menziesii ECM fungal community. These results suggest that, in terms of organic P and N acquisition, the most abundant ECM fungal species are not those with the highest relative activities. We also observed that less abundant ECM fungal species, particularly on P. menziesii, could have high relative AP and LA activities compared with other members of the same ECM community. Although the overall contribution of these less abundant fungi to proportional enzymatic activity is limited (i.e. when activity and abundance are multiplied), it does not appear that their root tip abundance is directly related to their relative organic P and N acquisition abilities. A similar lack of concordance between relative root tip abundance and enzyme activity was also observed by Rineau & Garbaye (2009) in non-limed European Picea abies and Fagus sylvatica stands. The consistency of this pattern across four ECM hosts in two different study systems, and with different types of symbiotic association, indicates that caution is needed when inferring links between individual ECM species abundance and their role in community enzymatic functioning. Although nutrient acquisition ability is likely to influence ECM fungal abundance patterns to some extent (Kimmel & Salant, 2006), the identification of additional biotic and abiotic factors that determine the relative abundance of individual ECM fungal species in both Alnus and other ECM host systems remains an important area of future research. Sources of root tip enzyme activity The exoenzyme assays used in this study involve excised ECM fungal root tips, which include both fungal and plant tissue, as well as root-associated prokaryotic communities. Given the mixed taxonomic nature of these structures, it is not possible to identify the specific sources of the enzymes being measured. We originally intended to assess the relative contribution of ECM fungi by comparing ECM and non-ECM root tip activities, but our sequencing of non-ECM root tips revealed that many were colonized by non-ECM fungi (data not shown). As ECM and non-ECM fungi can produce similar types and quantities of extracellular enzymes (Colpaert & Van Laere, 1996), we felt comparisons with non-ECM yet fungal-colonized tips were not an appropriate metric. As such, we did not try to formally partition the various contributions of fungal, plant and prokaryotic activity for the values obtained from the ECM colonized roots. New Phytologist (2014) 202: 287–296 www.newphytologist.com
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Jones et al. (2012) found that excised root tip potential enzymatic activity was overwhelmingly driven by the fungal symbiont, in spite of additional influential factors. Similarly, Cullings et al. (2008) found that the addition of antibiotics did not appreciably change the measured enzyme activities, which further supports the dominant contribution as being fungal in origin. Although we recognize that appropriate caution should be employed in interpreting this kind of assay data, we feel it does allow for effective relative comparisons of potential enzyme activities among both ECM fungal species and communities. Conclusion Collectively, our data indicate that the structure and functioning of ECM fungal communities may be linked, particularly in the context of tripartite interactions. In the Alnus host system, the presence of N-fixing bacteria appears to have shifted ECM fungal community activity towards a greater capacity for organic P acquisition and lower organic N acquisition. Given the major increases in N inputs into terrestrial ecosystems in recent decades (Vitousek et al., 1997), one might predict that the enzymatic activity of ECM fungi in non-Alnus systems may also shift towards greater P acquisition. Although ECM fungal functioning in many systems has yet to be characterized, Jones et al. (2012) showed that most ECM fungi in Pinus contorta stands exhibited limited plasticity in their organic P and N acquisition abilities in response to both short- and long-term N fertilization. This result suggests that the enzyme activity patterns observed here for the Alnus ECM fungal community reflect a long-term co-evolutionary interaction, rather than a plastic short-term response (Rochet et al., 2011). Although we believe enhanced P-acquiring abilities may be a key determinant of the atypical richness and specificity observed in Alnus ECM fungal communities, the ability to grow at significantly lower pH levels and higher nitrate concentrations may be important additional environmental filters limiting ECM fungal colonization in the Alnus rhizosphere. Determining the suite of traits that drive ecological specificity in ECM fungi and other plant–microbial symbioses remains an important challenge (Bruns et al., 2002), and additional studies examining the functioning of specialist and generalist microorganisms in other host systems will provide greater insight into this topic.
Acknowledgements We thank D. Hibbs for assistance with access to the field sites, J. Huggins and L. Bogar for help with sample collection, J. Talbot for guidance with enzyme assay design, and M.-A. Selosse and three anonymous reviewers for constructive comments on previous drafts of the manuscript. Funding was provided by the National Science Foundation (Grant no. DEB-1020735 to P.G.K.).
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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Molecular phylogenetic analysis of ectomycorrhizal (ECM) fungal root tip samples of Lactarius obscuratus using the maximum likelihood method based on the Jukes–Cantor model. Fig. S2 Nonmetric Multidimensional Scaling (NMDS) ordination of ectomycorrhizal (ECM) fungal community presence– absence on Alnus rubra and Pseudotsuga menziesii at Cascade Head and H.J. Andrews. New Phytologist (2014) 202: 287–296 www.newphytologist.com
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Fig. S3 Differences in potential enzyme activity between ectomycorrhizal (ECM) fungal communities at Cascade Head and H.J. Andrews in spring and summer.
Table S4 Ectomycorrhizal (ECM) fungal species present in at least 5% of cores sampled per host on Alnus rubra and Pseudotsuga menziesii at Cascade Head and H.J. Andrews
Table S1 Best database matches and accession numbers for representative query sequences of all ectomycorrhizal fungi identified at Cascade Head and H.J. Andrews
Table S5 Univariate ANOVAs of ectomycorrhizal (ECM) fungal community potential enzyme activity at Cascade Head and H.J. Andrews on A. rubra and P. menziesii
Table S2 Permutational MANOVA (Permanova) of ectomycorrhizal (ECM) fungal community composition based on presence–absence per sample (core), with plot nested in season, site and host, and based on Bray–Curtis distances
Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.
Table S3 Permutational MANOVA (Permanova) of ectomycorrhizal (ECM) fungal community composition based on presence–absence per plot and Bray–Curtis distances
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