Attributing Functions to Ectomycorrhizal Fungal Identities in Assemblages for Nitrogen Acquisition Under Stress

The ISME Journal (2014) 8, 321–330 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE Attributing functions to ectomycorrhizal fungal identities in assemblages for nitrogen acquisition under stress Rodica Pena and Andrea Polle Forest Botany and Tree Physiology, Georg-August Universita¨tGo¨ttingen, Bu¨sgenweg 2, Go¨ttingen, Germany Mycorrhizal fungi have a key role in nitrogen (N) cycling, particularly in boreal and temperate ecosystems. However, the significance of ectomycorrhizal fungal (EMF) diversity for this important ecosystem function is unknown. Here, EMF taxon-specific N uptake was analyzed via 15N isotope enrichment in complex root-associated assemblages and non-mycorrhizal root tips in controlled experiments. Specific 15N enrichment in ectomycorrhizas, which represents the N influx and export, as well as the exchange of 15N with the N pool of the root tip, was dependent on the fungal identity. Light or water deprivation revealed interspecific response diversity for N uptake. Partial taxon- specific N fluxes for ectomycorrhizas were assessed, and the benefits of EMF assemblages for plant N nutrition were estimated. We demonstrated that ectomycorrhizal assemblages provide advantages for inorganic N uptake compared with non-mycorrhizal roots under environmental constraints but not for unstressed plants. These benefits were realized via stress activation of distinct EMF taxa, which suggests significant functional diversity within EMF assemblages. We developed and validated a model that predicts net N flux into the plant based on taxon-specific 15N enrichment in ectomycorrhizal root tips. These results open a new avenue to characterize the functional traits of EMF taxa in complex communities. The ISME Journal (2014) 8, 321–330; doi:10.1038/ismej.2013.158; published online 12 September 2013 Subject Category: Microbe-microbe and microbe-host interactions Keywords: 15N labeling; beech (Fagus sylvatica); drought; modeling; mycorrhiza; shade Introduction Although it has been well established that EMF have key roles in plant nutrition, much less is The roots of most plant species are associated with known about the functions of distinct fungal taxa mycorrhizal fungi that mediate nutrient exchange within complex ectomycorrhizal assemblages for between the plants and soil and thus have a central nutrient acquisition and host supply. Ectomycorrhizal role in biogeochemical cycles (Finlay, 2008). In communities are usually composed of a diverse temperate and boreal forests, fungi that form flora consisting of several dominant and many ectomycorrhizas are the dominant symbiotic life infrequent EMF species (Bue´e et al., 2007; Courty form. Ectomycorrhizal fungi (EMF) encase colonized et al., 2010; Pena et al., 2010; Lang et al., 2011; root tips with a dense hyphal net, termed the Tedersoo et al., 2012a; Danielsen et al., 2013). EMF mantle, and forage the soil for nutrients by extend- community structures are strongly influenced by N ing extraradical hyphae or hyphal cords (Finlay, deposition (Lilleskov et al., 2011; Kjøller et al., 2008). As nitrogen (N) is a major limiting nutrient in 2012). Stable isotope studies have revealed that EMF many forest ecosystems (LeBauer and Treseder, species differ in their abilities to exploit different N 2008), the role of EMF in the N nutrition of trees sources (Hobbie and Ho¨gberg, 2012). Furthermore, has received considerable attention (Hobbie and in situ ectomycorrhizal communities exhibit strong Hobbie, 2008; Hobbie and Ho¨gberg, 2012). In temporal differences in the capability of different addition to N delivery, recent studies have suggested EMF taxa to access litter-derived N (Pena et al., that EMF may also limit N transfer to host trees 2013a). The experimental manipulation of EMF under N-limiting conditions (Na¨sholm et al., 2013). diversity has shown context-dependent effects for fungal mixtures on plant biomass production and N nutrition (Chu-Chou and Grace, 1985; Jonsson et al., Correspondence: A Polle, Forest Botany and Tree Physiology, 2001). As the mechanistic concepts that explain Georg-August Universita¨tGo¨ttingen, Bu¨sgenweg 2, 37077 the interactions between different EMF taxa in Go¨ttingen, Germany. E-mail: [email protected] complex assemblages are still missing, the func- Received 28 April 2013; revised 25 July 2013; accepted 8 August tional relevance of EMF identities for tree nutrition 2013; published online 12 September 2013 remains enigmatic. Elucidating functional diversity Response diversity of ectomycorrhizal fungi R Pena and A Polle 322 is important for understanding the role of ectomy- (Pena et al., 2013b). Ah horizon soil (20 cm depth) corrhizal fungi in biogeochemical cycles in a collected in the Tuttlingen beech forest (latitude fluctuating environment. 471590N, longitude 81450E) was used. The germi- Our study aimed to attribute functions for N nated seedlings were maintained in a greenhouse acquisition to ectomycorrhizal species identities in under ambient conditions (20 1C, 55% air humidity) root-associated assemblages and uncover taxon- with additional light to achieve a 16-h photoperiod specific responses to environmental stress factors. with 200 mmol PAR m À 2 s À 1 at plant height (lamps We used young beech (Fagus sylvatica L.) trees, series 3071, Schuch, Worms, Germany). After 4 which are the major tree species of the natural months, eight seedlings per treatment were evalu- vegetation in Central European temperate forests ated for their mycorrhizal status showing that (Ellenberg and Strutt, 2009). The current beech roots of seedlings in untreated soil were 40±4% forest distribution range is endangered as a result colonized by EMF, whereas seedlings in sterilized of drought stress because of climate change (Weber soil were non-mycorrhizal. A total of 120 mycor- et al., 2013). Beech trees are tolerant of deep shade rhizal and 120 non-mycorrhizal beech seedlings in the youth phase (Ellenberg and Strutt, 2009); were transplanted without adherent soil individu- however, shade-induced carbon limitations have a ally into 660 ml pots with a sand-peat mixture negative impact on EMF colonization (Druebert and supplied daily with 56 ml of nutrient solution þ et al., 2009). Here, we conducted controlled experi- containing 0.4 mM NH4 as the sole N source. ments with beech seedlings cultivated in natural This concentration was chosen because it was forest soil to develop characteristic EMF communities. similar to that in forest soil of the Tuttlingen site The mycorrhizal trees were subsequently grown in (Dannenmann et al., 2009), well above Km values for þ sand to permit analysis of intact root systems and NH4 uptake of various EMF (0.005–0.25 mM; þ supplied with ammonium (NH4 ) concentrations Jongbloed et al., 1991; Eltrop and Marschner, 1996) similar to those found in beech forest soils (median and because preceding analysis with attached þ À 1 þ 0.5 mmol NH4 kg soil; range: 0.05–2 mmol NH4 beech roots at the Tuttlingen site showed saturation À 1 þ kg soil; Gessler et al., 2005; Go¨ransson et al., of NH4 uptake at concentrations above 0.05 mM 2006; Dannenmann et al., 2009; Andreasson et al., (Gessler et al., 2005). The seedlings were grown 2012). Subsets of the plants were exposed to either in full light (200 mmol PAR m À 2 s À 1) or in the full light or shade according to the characteristic shade (35–40 mmol PAR m À 2 s À 1 at plant height). light climate in beech forests (median: 150 mmol The light climate was chosen according to the PAR m À 2 s À 1, range 25–255 mmol PAR m À 2 s À 1; conditions in thinned and unthinned beech plots Kreuzwieser et al., 1997; Lemoine et al., 2002; in the Tuttlingen forest with mean seasonal light Mayer et al., 2002; Fotelli et al., 2003; Gessler levels of 176 and 25 mmol PAR m À 2 s À 1, respectively et al., 2005; Hertl et al., 2012). Light and shade (Gessler et al., 2005). After 2 months, the irrigation treatments were combined with sufficient irrigation solution was reduced to 37% for half of the plants or water shortage to mimic typical environmental grown under each light regime. After 16 days, when stresses. N acquisition was measured after the the well-irrigated plants had a predawn leaf water application of 15N in root tips associated with potential of À 0.36±0.02 MPa and those subjected to distinct EMF species and non-mycorrhizal root tips. a limited water supply had a predawn leaf potential We tested the hypotheses that (i) ectomycorrhizal of À 1.34±0.06 MPa, the plants were harvested assemblages show taxon-specific differences for (Pena et al., 2013b). During the last 3 days before þ NH4 acquisition and (ii) environmental stress harvest, each beech seedling received a total of results in functional shifts in EMF species for N 1.864 mg of N in a solution of either non-labeled 15 acquisition. Partial fluxes for EMF-associated root NH4Cl or NH4Cl (99 atom %, Cambridge Isotope tips were assessed with whole-plant N uptake Laboratories, Inc., Hampshire, UK). Biomass and for mycorrhizal and non-mycorrhizal plants. whole plant 15N data were determined (Pena et al., We provide evidence that the taxon-specific 15N 2013b). Ten 15N labeled and three non-labeled plants enrichments in EMF-associated root tips can be used per treatment were randomly selected and used for to predict net N flux into the host plant. These the analyses. results provide a basis for testing functional redun- dancy and response diversity of EMF assemblages in future field studies. EMF identification and quantification For each growth regimen, the whole root system of 10 beech seedlings per treatment was inspected Materials and methods using a binocular microscope (Leica M205 FA, Leica Microsystems, Wetzlar, Germany). Within each Plant cultivation and experimental treatments sample, the root tips were assigned to one of the Fungicide-treated beech nuts (Fagus sylvatica L., following fractions: vital ectomycorrhizal (EM), vital provenance: Forstsaatgutstelle Oerrel, Niedersach- non-ectomycorrhizal (NM), dead ectomycorrhizal sen, Germany) were grown in sterilized or untreated (DM) and dead non-ectomycorrhizal (DR).

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