Title: The fungal perspective of arbuscular mycorrhizal colonization in ‘nonmycorrhizal’ plants
Abstract: New PhytologistVolume 205, Issue 4 p. 1399-1403 LettersFree Access The fungal perspective of arbuscular mycorrhizal colonization in ‘nonmycorrhizal’ plants Ylva Lekberg, Corresponding Author Ylva Lekberg MPG Ranch, 1001 S. Higgins Ave, Missoula, MT, 59801 USA Department for Ecosystem and Conservation Sciences, University of Montana, Missoula, MT, 59812 USA(Author for correspondence: tel +1 406 396 6159; email [email protected])Search for more papers by this authorSøren Rosendahl, Søren Rosendahl Department of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, DenmarkSearch for more papers by this authorPål Axel Olsson, Pål Axel Olsson Biodiversity, Department of Biology, Lund University, Ecology Building, SE-223 62 Lund, SwedenSearch for more papers by this author Ylva Lekberg, Corresponding Author Ylva Lekberg MPG Ranch, 1001 S. Higgins Ave, Missoula, MT, 59801 USA Department for Ecosystem and Conservation Sciences, University of Montana, Missoula, MT, 59812 USA(Author for correspondence: tel +1 406 396 6159; email [email protected])Search for more papers by this authorSøren Rosendahl, Søren Rosendahl Department of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, DenmarkSearch for more papers by this authorPål Axel Olsson, Pål Axel Olsson Biodiversity, Department of Biology, Lund University, Ecology Building, SE-223 62 Lund, SwedenSearch for more papers by this author First published: 08 October 2014 https://doi.org/10.1111/nph.13118Citations: 27AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Arbuscular mycorrhiza (AM) is arguably the most abundant symbiosis on Earth. It involves fungi in the Glomeromycota and c. 70% of vascular plants (Brundrett, 2009), in which the fungal partner aids in nutrient uptake, pathogen protection and possibly other services in exchange for plant carbon (C) (Smith & Read, 2008). A smaller, but not insignificant, number of plant species are considered to be nonmycorrhizal, especially those within the families Amaranthaceae, Chenopodiaceae, Carophyllaceae and Brassicaceae (Brundrett, 2009). However, fungal colonization that resembles AM is often observed in these ‘nonmycorrhizal’ plants when they co-occur with mycorrhizal plants (Hirrel et al., 1978; Miller et al., 1983), although arbuscules are rarely if ever present (Meney et al., 1993; Muthukumar et al., 1996; Sengupta & Chaudhuri, 2002). Because arbuscules are the structures where AM fungi deliver phosphorus (P) to plants (Smith & Read, 2008), and since the hyphal and vesicular colonization is often observed in old or dying roots (Brundrett, 2004), the function of this symbiosis has been questioned (Hirrel et al., 1978; Koide & Schreiner, 1992; Brundrett, 2004). Nonetheless, responses to AM fungal inoculations by these plants range from negative (Allen et al., 1989) to positive (Williams et al., 1974), which suggests that mycorrhizal associations are not always asymptomatic, and that the distinction between mycorrhizal and nonmycorrhizal plants is more subtle than sometimes believed. Acknowledging its limitation, we will use the term ‘nonhost’ here to refer to plants that are thought to be nonmycorrhizal but often appear to be colonized by AM fungi. AM fungal colonization of nonhost plants has been quantified for decades (Hirrel et al., 1978), but we know surprisingly little about what the benefits might be for the fungus. For example, if colonization mainly occurs in old or dying nonhost roots (Brundrett, 2004), does this represent an important P source for the fungus to be allocated to other, better host plants, similar to what has been observed when killing or defoliating highly mycorrhizal plants (Eason et al., 1991; Mikkelsen et al., 2008)? Also, does the C stored in vesicles come from the nonhost, or does the fungus simply use the roots as shelter and C storage (Lekberg et al., 2010)? And finally, because these plants may differ from mycorrhizal plants in root anatomy and chemistry (Koide & Schreiner, 1992), are nonhost plants colonized by a broad array of AM fungi or restricted to a narrow lineage, similar to what has been shown for mycoheterotrophic plants (Bidartondo et al., 2002)? Here we set out to explore the latter two questions using a putative nonhost plant, Dianthus deltoides in the Caryophyllaceae (Wang & Qui, 2006), which co-occurs with mycorrhizal plants in a Danish coastal grassland. In June, 2007, we sampled 16 Dianthus plants in a 50 m × 100 m area for assessments of AM colonization and fungal community composition. We also sampled 16 Hypochoeris radicata plants (a highly mycorrhizal species) to allow for comparisons between Dianthus and a typical host plant. Plants were sampled in a pairwise manner (Dianthus and Hypochoeris plants were no more than > 30 cm apart) to reduce confounding effects of AM fungal spatial patterns, which have been documented at this site previously (Rosendahl & Stukenbrock, 2004). We assessed AM colonization on trypan blue stained roots using the gridline intersect method on at least 50 intersects (McGonigle et al., 1990), and extracted and amplified DNA from roots using the same method as in Lekberg et al. (2012). Due to the visually lower AM fungal abundance in Dianthus roots, we extracted DNA from eight 2-cm root pieces per plant compared with eight 0.5-cm pieces per Hypochoeris plant. Briefly, we targeted the large ribosomal subunit (LSU) and amplified DNA using a nested PCR with the eukaryotic primers 0061 and NDL22 followed by FLR3-FLR4 (Van Tuinen et al., 1998). Because we extracted DNA from short root fragments, all positive PCR products were sequenced directly without cloning (Macrogen, Seoul, Korea). High quality sequences were aligned with published sequences obtained from the same grassland and elsewhere (Rosendahl & Stukenbrock, 2004; Stukenbrock & Rosendahl, 2005; Lekberg et al., 2012). We considered sequences a match with previous operational taxonomic units (OTUs) if they clustered within their monophyletic clades (see Supporting Information Table S1 for original OTU names, updated nomenclature and published accession numbers). We used a chi-square test (with Yates correction; http://statpages.org/) and a 2 (plant species) × 10 (OTU) contingency table to test for differences in AM fungal community composition between Dianthus and Hypochoeris based on presence/absence of OTUs in each plant. To verify that AM fungi were responsible for the observed root colonization, and to assess if Dianthus allocates C to AM fungi, we used neutral lipid fatty acid (NLFA) stable isotope probing (Olsson et al., 2005) and conducted a 13CO2 pulse-chase labeling on seven Dianthus individuals in July 2008. Given that this sampling addressed a separate question from the sampling in 2007, the different sampling times should present no problem. The labeling and post-labeling processing was identical to that described in Lekberg et al. (2012), and results from the Dianthus plants were compared with seven Hypochoeris individuals from the study by Lekberg et al. (2012). These Hypochoeris plants were from the same 50 m × 100 m area, and had an identical labeling time (three consecutive days starting 21 July for 2 h d−1) and chase period (3 d after the last labeling). All plants were at least 5 m away from each other to eliminate any risk of 13C cross-contamination. Carbon-13 (13C) concentration (expressed as δPDB values) was quantified at harvest in shoots and roots to assess C-assimilation and allocation belowground, and one nonlabelled plant per species was harvested for background 13C concentration. Lipids were extracted from freeze-dried fine roots (n = 8 per species) and analyzed as described earlier (van Aarle & Olsson, 2003; Lekberg et al., 2012). In order to assess the C-allocation specifically to AM fungi, we measured the 13C concentration in the AM fungal storage lipid NLFA 16:1ω5 in all labeled plant roots by compound specific isotope ratio mass spectrometry (Olsson et al., 2005). To control for differences in overall allocation belowground between the two plant species, we also compared 13C concentration in NLFA 16:0 (occurs both in plants and AM fungi) and in NLFA 18:2ω6,9 (in plant roots but not in AM fungi). Similar to previous studies involving nonhost plants (Hildebrandt et al., 2001), Dianthus roots were colonized by hyphae and vesicles but rarely by arbuscules (two Dianthus plants had < 5% arbuscular colonization), and overall AM colonization was substantially lower (P < 0.001, t-test) than in Hypochoeris plants (Fig. 1). This was supported by large differences (P < 0.001, t-test on loge-transformed values) in 16:1ω5 concentrations between Dianthus (11.1 ± 5.7 nmol g−1 root, mean ± standard error (SE)) and Hypochoeris roots (536 ± 180 nmol g−1 root). We do not know if Dianthus can form AM in the absence of mycorrhizal plants (sensu Atriplex confertifolia in Miller et al., 1983), but the colonization of field plants suggests that the nearly nonhost status of this plant is not due to cell wall structures or antifungal compounds that deter hyphal growth and penetration. Lack of signaling may explain the low AM colonization (discussed in Koide & Schreiner, 1992) and this could be tested in controlled inoculation experiments with Dianthus grown with and without good host plants. Figure 1Open in figure viewerPowerPoint Overall arbuscular mycorrhiza (AM) colonization (dark gray), arbuscular (medium gray) and vesicular (light gray) colonization of Hypchoeris radicata and Dianthus deltoides in a Danish coastal grassland and micrographs of stained roots from the same site (inserted pictures). Reflecting the lower AM colonization, only three Dianthus plants had roots with detectable NLFA 16:1ω5, which clearly limits our ability to test if Dianthus allocates C to AM fungi. Due to this, and because of the large variation among Dianthus replicates, we chose not to analyze the data statistically but instead to plot individual values for visual comparisons against mean values for Hypochoeris (Fig. 2). The 13C concentrations in NLFA 16:1ω5 in the three Dianthus roots are visually lower than the mean values for Hypochoeris, and because 13C concentration measures recently allocated C per unit fungal storage lipid (as indicated by NLFA 16:1ω5), these lower values are not due to a lower AM fungal abundance in Dianthus. Nor are potential differences driven by plant size and 13C assimilation because they were comparable between the two species (Table S2), as was the C-allocation to roots (indicated by similar 13C concentration in the plant specific NLFA 18:2ω6,9, Fig. 2). It is interesting to note, however, that NLFA 16:1ω5 was enriched in all Dianthus samples compared to the unlabeled plants (Fig. 2), suggesting that at least some C in the AM fungi came from Dianthus. This enrichment cannot be explained by C-fractionation during the transfer of C from plant to AM fungi, because the difference in natural abundance of 13C between the two symbionts is < 2‰ (Staddon et al., 1999), and the least enriched sample showed an enrichment of > 14‰. The 13C concentration in NLFA 16:1ω5 varied substantially within Dianthus for unknown reasons, and the low enrichment in two of the samples may simply have resulted from an assimilation of apoplastic C without any active allocation from – or real cost to – the plant (see discussions in Lekberg & Koide, 2014). The sample with the higher enrichment may suggest a more active symbiosis, but careful pairing of costs and benefits is needed to assess that. We want to stress that more samples are required for robust conclusions, and we urge readers to view our results as preliminary. However, our findings agree with Allen & Allen (1990) who showed that nonhost plants contribute at least some C for spore production, although spore numbers were much lower than under a good host. As in Allen & Allen (1990) we observed a higher C investment to AM fungi by the good host, because Hypochoeris appeared to allocate more C per unit fungal biomass and also supported a much greater fungal biomass than Dianthus. Combined with the higher arbuscular colonization in Hypochoeris, our field survey support previous findings of a balanced resource trade in the AM symbiosis, in which symbionts that invest more C receive more resources in return (Lekberg et al., 2010; Hammer et al., 2011). Our measure of AM colonization and its different components also show that these measurements can be informative. Figure 2Open in figure viewerPowerPoint Carbon-13 (13C)-concentration as indicated by δ13C values for three neutral lipid fatty acids: 16:1ω5 (specific to arbuscular mycorrhiza (AM) fungi), 16:0 (common in all types of organisms) and 18:2ω6,9 (common in plants, but not in AM fungi) in the seven Hypochoeris (gray squares, average ± standard error (SE)) and the three Dianthus samples that contained sufficient neutral lipid fatty acid (NLFA) 16:1ω5 concentrations (black diamonds). The δ13C values in roots of unlabeled Hypochoeris and Dianthus were −28.4 and −25.3, respectively (dashed line show the average of the two values). Nine of the 16 Dianthus plants and all 16 Hypochoeris plants yielded at least one PCR product that was sequenced successfully (average PCR success was 12% and 59% of root segments, respectively). We found a total of four OTUs in Dianthus roots and eight OTUs in Hypochoeris roots. The two plant species harbored different AM fungal communities (P = 0.04); Dianthus was dominated by an OTU that clustered with Funneliformis mosseae, whereas Cluster D fungi dominated in Hypochoeris roots (Fig. 3; Table S1). The high abundance of Cluster D in Hypochoeris was not surprising given that it is an ubiquitous fungus at this site (Rosendahl & Stukenbrock, 2004; Stukenbrock & Rosendahl, 2005), but the dominance of F. mosseae in Dianthus was unexpected as it is relatively rare and was only detected in c. 10% of Hypochoeris plants in a large survey (Lekberg et al., 2012). This nonrandom host association is particularly interesting given that no difference in AM fungal communities has been documented among highly colonized plants in this grassland previously (Stukenbrock & Rosendahl, 2005). We deliberately choose the term ‘nonrandom host association’ in favor of ‘host preference’, because the higher abundance of F. mosseae in Dianthus may have little to do with a preference for Dianthus. On the contrary, F. mosseae may very well prefer other, better hosts, but could be limited in those root systems by abundant OTUs that may be superior competitors. There are also clear differences in life history strategies between the fungi that dominate in the two plant species that could contribute to the compositional differences observed. For example, Cluster D form large mycelia (Rosendahl & Stukenbrock, 2004) and appear never to sporulate (Lekberg & Rosendahl, personal observations), whereas F. mosseae is considered a ruderal species that sporulates frequently (Sykorova et al., 2007). Because AM fungi may differ in their ability to acquire C from hosts (Pearson & Jakobsen, 1993), it is also possible that host quality could be an additional, less recognized, driver of community assembly. Indeed, the distribution of Dianthus and another putative nonhost plant, Carex arenaria (Cyperaceae) correlated significantly with shifts in OTU compositions in a previous large-scale survey in this grassland (Lekberg et al., 2012), suggesting that our more limited sampling reflects plant–AM fungal community interactions within this grassland. Figure 3Open in figure viewerPowerPoint The distribution of sequence types differed (P = 0.04) between Hypochoeris radicata and Dianthus deltoides. The dominant sequence type in Dianthus (Funneliformis mosseae) was not found in Hypochoeris. Sequence names are adopted from Rosendahl & Stukenbrock (2004), Stukenbrock & Rosendahl (2005) and Lekberg et al. (2012) with some updated nomenclature based on Redecker et al. (2013). Work on mycoheterotrophic plants has lead to suggestions that the degree of host specificity in AM is greater in plants where only the plant receives benefits from the association (Brundrett, 2004). For example, nonphotosynthetic plants are predominately colonized by Glomus group A fungi (Merckx et al., 2012) and Bidartondo et al. (2002) suggested that these fungi may mediate inter-plant C transfer. Perhaps co-incidental, but Dianthus was also colonized by Glomus group A fungi, and it is tempting to speculate about other similarities between nonhost plants and mycoheterotrophs; neither plant group likely delivers much C to the fungus, and arbuscules are seldom observed. Whether or not these similarities mean anything is uncertain, and we do not mean to imply that Dianthus receives C from the fungus. By combining microscopic examinations, fatty acid analysis and molecular identification, our results indicate that the putative nonmycorrhizal plant Dianthus deltiodes is colonized by a particular subset of AM fungi in this Danish grassland. This suggests that the traditional distinction between host and nonhost is not very useful and that mycorrhizal associations are more nuanced than this dichotomy indicates. Brundrett (2009) argued that colonization by AM fungi in typical nonhost plants should be referred to as Glomeromycotan fungus colonization (GFC) as opposed to AM given that the ecological significance of these interactions are likely low. Indeed, the absence of arbuscules in most Dianthus plants here indicates that resource delivery from AM fungi to the plants is probably minimal. However, if the intraradical colonization is high, AM fungi may start competing with putative pathogens for space and resources and result in some pathogen protection (Sikes et al., 2009). From the fungal perspective, these associations may be more important, and our results suggest that some C can be delivered from Dianthus to AM fungi. Also, because AM fungi can store a substantial amount of the C it acquires from one host in other neighboring roots (Robinson & Fitter, 1999), it is possible that F. mosseae largely uses Dianthus roots as a refuge to store C that it gains from spotty colonization of better hosts. Colonization for the sole purpose of acquiring nutrients from dying and decomposing roots (Brundrett, 2004) seems unlikely here, because Dianthus roots all appeared healthy. In summary, while most research on the mycorrhizal status of nonhost plants was published decades ago, modern DNA- and isotope-based tools allow us to probe the physiological and ecological significance of this specific symbiosis, which can increase our understanding of plant–AM fungal interactions in general. It is our hope that this small study will inspire others to address these questions elsewhere to substantiate (or refute) findings presented here. Acknowledgements The authors thank Roger Koide, Philip Ramsey, Lauren Waller and three anonymous reviewers for their constructive comments on the manuscript. Y.L. is grateful to MPG Operations and P.A.O. to the Swedish Research Council for funding. Supporting Information 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. 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Soil Science Society of America Journal 38: 962– 965. Wiley Online LibraryWeb of Science®Google Scholar Citing Literature Volume205, Issue4Special Issue: Ecology and evolution of mycorrhizasMarch 2015Pages 1399-1403 This article also appears in:Ecology and evolution of mycorrhizas FiguresReferencesRelatedInformation