Mycorrhizal Network and Symbiotic N-Fixer Jointly Enhance the Interplant Nitrogen Sharing.

Nitrogen (N) exchange mediated through common mycorrhizal networks (CMNs) was investigated in pure and mixed systems of Casuarina cunninghamiana and Eucalyptus maculata, and Glycine max (soybean) and Sorghum bicolour (sorghum). Both 15N labeling and 15N natural abundance (δ 15N) studies were performed. Seeds of all four species were aseptically germinated with or without mycorrhizal fungi or N2-fixing bacteria on agar media in petri dishes. Seedlings were transplanted into three-compartment growth units and paired. 37 μm diameter nylon mesh, RainSaver crystals (high water holding capacity) and N-serve (nitrification inhibitor) prevented direct root contact, soil nutrient flow with water and nitrification, respectively. At harvest, none of the controls was mycorrhizal or nodulated, while all originally non-mycorrhizal seedlings were colonised. Mycorrhizal hyphae penetration through the nylon mesh was directly observed and demonstrated with an Environmental Scanning Electron Microscope (ESEM). Colonisation of roots as high as 80% confirmed that common ectomycorrhizal or arbuscular mycorrhizal networks were established between pairs in all combinations. Mycorrhization had significant effects on biomass production in both N2-fixing plants (Casuarina, soybean) and non-N2-fixing ones (Eucalyptus, Sorghum). Dry matter production was highest in both partners when N2-fixing plants were mycorrhizal and nodulated. However, mycorrhization had little impact on N accumulation in eucalypts, but had a major effect in casuarinas, despite eucalypts having nearly double the colonisation rate. Biomass was positively correlated with tissue N content in both species. The nodulated mycorrhizal casuarinas and their companion mycorrhizal eucalypts had the highest tissue N accumulation. Both biomass and total N in all N-receivers equalled those in N-donors, especially when nodulated casuarinas were N-receivers. The above trends were generally true for soybean and sorghum pairs. In addition, δ15N values were negative in nodulated casuarinas, but positive in nodulated soybeans. Biological nitrogen fixation (BNF) contributed up to 50% and 40% of N in nodulated mycorrhizal casuarinas and soybeans, respectively. From both 15N labelling experiments and δ15N analyses it was established that N-transfer occurred bidirectionally (two-way) between Casuarina and Eucalyptus, and between soybean and Sorghum. The percentages and amounts of N transferred, and the % of N in the receiver derived from the transfer (%NDFT) were generally significantly higher in the nodulated/mycorrhizal pairs than in the nonnodulated/ mycorrhizal pairs. This occurred regardless of whether the nodulated N2-fixing plants were 'N-donors' or 'N-receivers'. However, the %NDFT was always on the same scale regardless of the direction of N-transfer. The % and amount of N-transfer were also significant from non-N2-fixing plants to nodulated N2-fixing plants (with up to 50% biological N2-fixation) rather than the reverse. Significantly higher bidirectional and net N-transfer were also found between the sole mycorrhizal and the nodulated/mycorrhizal pairs. These results indicated a net gain in N by N2-fixing plants, but not by non-N2-fixing ones. The similar N transferred to non-N2-fixing plants and to N2-fixing ones in the sole mycorrhizal pairs indicated that two-way N-transfer could occur naturally between any mycorrhizal plants, regardless of whether they were N2-fixing plants or non-N2-fixing ones, and that N resources could equally be reallocated between plants through mycorrhizae. The significantly greater intensity of bidirectional N-transfer in the nodulated mycorrhizal pairs showed that more substantial amounts of N could be shuttled between plants because of a generally greater physiological and ecological N demand in low-external-N-input conditions. These results therefore suggest that N2-fixing capacity might not be a prerequisite for, but might affect the intensity of, this two-way N-transfer. In addition to accessing N from soils directly by roots, the experiments suggest that N2-fixing plants have two further strategies, N2-fixation and mycorrhization, to satisfy their high N-demand, while mycorrhization alone can meet the needs for relatively low N-demand by non-N2-fixing plants. Two 'mycocentric' N-transfer mechanisms are postulated to account for these differences. It seems that any plant that gives more N than it receives is an 'N-donor', while the opposite is true for an 'N-receiver'. If these mechanisms operate as these experiments have demonstrated and prove to be widespread, ideas about mycorrhiza-mediated N exchange and cycling in both agricultural and natural ecosystems may have to be re-evaluated, and concepts about nutrient cycling and energy exchange in plant communities may also have to be reformulated. Bidirectional N-transfer certainly has important implications for the nitrogen economy of N2-fixation-based agricultural and natural ecosystems. In such ecosystems, the magnitude of mycorrhiza-mediated N-transfer and N movement seems to be determined by the dynamic four-way interactions between plant roots, mycorrhizal fungi, N2-fixing bacteria, and N resource availability and requirements.

Almost all land plant species form a symbiosis with mycorrhizal fungi. These soil fungi provide nutrients and other services to plants in return for plant carbohydrates. The recent application of microbial metagenomics, metatranscriptomics, and metabolomics to plants and their immediate surroundings confirms the key role of mycorrhizal fungi, rhizosphere bacteria and fungi, and suggests a world of hitherto undiscovered interactions (van der Heijden et al., this issue, pp. 1406–1423). This novel knowledge is leading to a paradigm-shifting view: plants cannot be considered as isolated individuals any more, but as metaorganisms, or holobionts (Hacquard & Schadt, this issue, pp. 1424– 1430) encompassing an active microbial community re-programming host physiology (see Pozo et al., this issue, pp. 1431–1436). This bears tremendous implications for plant ecophysiology and evolution, plant breeding, crop management and sustainable ecosystem management.

Both endophytic and mycorrhizal fungi interact with plants to form symbiosis in which the fungal partners rely on, and sometimes compete for, carbon (C) sources from their hosts. Changes in photosynthesis in host plants caused by atmospheric carbon dioxide (CO2) enrichment may, therefore, influence those mutualistic interactions, potentially modifying plant nutrient acquisition and interactions with other coexisting plant species. However, few studies have so far examined the interactive controls of endophytes and mycorrhizae over plant responses to atmospheric CO2enrichment. UsingFestuca arundinaceaSchreb andPlantago lanceolataL. as model plants, we examined the effects of elevated CO2on mycorrhizae and endophyte (Neotyphodium coenophialum)and plant nitrogen (N) acquisition in two microcosm experiments, and determined whether and how mycorrhizae and endophytes mediate interactions between their host plant species. Endophyte‐free and endophyte‐infectedF. arundinaceavarieties,P. lanceolataL., and their combination with or without mycorrhizal inocula were grown under ambient (400 μmol mol−1) and elevated CO2(ambient + 330 μmol mol−1). A15N isotope tracer was used to quantify the mycorrhiza‐mediated plant acquisition of N from soil. Elevated CO2stimulated the growth ofP. lanceolatagreater thanF. arundinacea, increasing the shoot biomass ratio ofP. lanceolatatoF. arundinaceain all the mixtures. Elevated CO2also increased mycorrhizal root colonization ofP. lanceolata, but had no impact on that ofF. arundinacea. Mycorrhizae increased the shoot biomass ratio ofP. lanceolatatoF. arundinaceaunder elevated CO2. In the absence of endophytes, both elevated CO2and mycorrhizae enhanced15N and total N uptake ofP. lanceolatabut had either no or even negative effects on N acquisition ofF. arundinacea, altering N distribution between these two species in the mixture. The presence of endophytes inF. arundinacea, however, reduced the CO2effect on N acquisition inP. lanceolata, although it did not affect growth responses of their host plants to elevated CO2. These results suggest that mycorrhizal fungi and endophytes might interactively affect the responses of their host plants and their coexisting species to elevated CO2.

Summary 1. Almost all plants are engaged in symbiotic relationships with mycorrhizal fungi. These soil fungi can promote plant growth by supplying limiting nutrients to plant roots in return for plant assimilates. 2. Many mycorrhizal fungi are not host specific and one fungal individual can colonize and interconnect a considerable number of plants. The existence of these so‐called mycorrhizal networks implies that fungi have the potential to facilitate growth of other plants and distribute resources among plants irrespective of their size, status or identity. In this paper, we explore the significance of mycorrhizal fungal networks for individual plants and for plant communities. 3. We address the following questions: (i) are all plant species benefitting from mycorrhizal networks, (ii) is benefit dependent on the size or age of a plant, (iii) is fungal support related to the relative dominance of plants in a community, (iv) are there host dependent barriers and physiological constraints for support and (v) what is the impact of mycorrhizal networks on plant–plant interactions and plant community dynamics? Moreover, using a review of published studies, we test whether mycorrhizal networks facilitate growth of small seedlings that establish between or near larger plants. 4. We found 60 cases where seedling species were grown together with larger plants with or without mycorrhizal fungal networks. Mycorrhizal networks promoted seedling growth in 48% of the cases (for 21 seedling species), while negative effects (25%) and no effects (27%) were also common. Seedlings associating with ectomycorrhizal fungi benefitted in the majority of the cases while effects on seedlings associating with arbuscular mycorrhizal fungi were more variable. Thus, the facilitative effects of mycorrhizal fungal networks depend on seedling species identity, mycorrhizal identity, plant species combinations and study system. We present a number of hypothetical scenarios that can explain the results based on cost–benefit relationship of individual members in a network. 5. Synthesis. Overall, this review shows that mycorrhizal networks play a key role in plant communities by facilitating and influencing seedling establishment, by altering plant–plant interactions and by supplying and recycling nutrients.

Chapter 18 - Mycorrhizal Networks and Forest Resilience to Drought

Plants typically interact with multiple, co‐occurring symbionts, including arbuscular mycorrhizal (AM) fungi which can form networks, connecting neighbouring plants. A characteristic aspect of the mycorrhizal symbiosis is the bidirectional exchange of nutrients between host plants and fungal partners. Concurrent interactions with competing organisms such as aphids or potato cyst nematodes (PCN) can disrupt the carbon‐for‐nutrient exchange between plants and AM fungi. However, the role of mycorrhizal networks (MNs) in mediating these interactions remains unclear. Using isotope tracing in multi‐plant experimental systems, we investigated the movement of plant photosynthates and fungal‐acquired soil phosphorus through MNs and the interactive effects of PCN infection on this. We found evidence of preferential allocation of fungal‐acquired phosphorus to plants that were not infected by PCN compared with infected neighbours. Contrary to previous findings using single plants, we did not detect a PCN‐induced reduction in the amounts of plant carbon delivered to AM fungi in multi‐plant systems. However, the MN(s) moved plant‐fixed carbon away from PCN‐infected host plants, regardless of the PCN infection status of the neighbouring plant host. Our work highlights the responsiveness of MNs to interactions with below‐ground organisms. It also strengthens the argument for a more mycocentric view of AM–plant symbioses. Experimental designs of increasing ecological complexity are needed for a more comprehensive understanding of the carbon‐for‐nutrient dynamics in AM fungi–plant networks. This will, in turn, elucidate the role of AM fungi in terrestrial carbon cycling and their function in agricultural systems. Read the free Plain Language Summary for this article on the Journal blog.

Unravelling potassium nutrition in ectomycorrhizal associations

Eupatorium adenophorum is an alien species that threatens community stability and diversity in karst areas. Arbuscular mycorrhizal (AM) fungi form interconnected mycorrhizal network, connecting adjacent plants and plant species. How mycorrhizal networks affect the competition for nutrients between invasive and native plants in karst habitat remains unclear at present. An experiment was conducted using a compartmental growing device, which was composed of two planting compartments (for the invasive E. adenophorum or native Artemisia annua) and a competitive compartment (for the interconnected mycorrhizal network). The experiment contained mycorrhizal fungus treatments, with AM fungi (M+) and without AM fungi (M−) using the species Claroideoglomus etunicatum, and the nutrient utilization treatments using nylon mesh to interconnective mycorrhizal networks, including common utilization (Cu), single utilization (Su), and non-utilization (Nu). The results showed as follows: AM fungi differentially increased biomass, nitrogen (N) acquisition and phosphorus (P) acquisition and significantly reduced N/P ratio of the invasive E. adenophorum and native A. annua under Cu, Su, and Nu conditions. Additionally, the biomass, N acquisition and P acquisition of E. adenophorum was greater than A. annua and the N/P ratio of E. adenophorum was significantly lower than A. annua under Cu condition, which AM fungi promoted the accumulation of biomass, N and P for E. adenophorum and A. annua, and E. adenophorum experienced a greater reduction of P limitation than A. annua via the interconnected mycorrhizal network. In conclusion, we suggest that AM fungi endow invasive plants greater alleviation of P limitation and enhancement of nutrient competition than native plants via mycorrhizal network in low-P karst soil.

The symbiotic relationship between mycorrhizal fungi and plants predates the origin of roots and has played a key role in shaping ecosystems for hundreds of millions of years. In associating with multiple plants simultaneously, mycorrhizal fungi can form complex below‐ground networks that directly—and indirectly—influence plant communities, plant and soil resource dynamics, and broader ecosystem processes. Research has provided increasing insight into the structure and function of these networks, including the movement and exchange of resources between symbionts, the mechanisms governing fungal and plant community assembly, and their potential applications in land management. As public interest in mycorrhizal networks has grown, so too have calls within the scientific community for greater clarity regarding their ecological functionality and broader significance. This Special Focus brings together research that advances our understanding of these networks from multiple perspectives. Contributions explore the hierarchical complexity of fungal‐plant associations, the ecological and functional implications of mycorrhizal selectivity, the resource exchange dynamics, and their relevance in applied contexts, such as agriculture. By synthesising emerging evidence, this collection highlights key advances while also identifying unresolved questions and the future research directions necessary for disentangling the ecological roles of mycorrhizal networks. Read the free Plain Language Summary for this article on the Journal blog.

The non-reducing disaccharide trehalose (a-D-glucopyranosyl-1,1-a-D-glucopyranoside) is widespread in nature, but is normally not present in higher plants. With respect to plant-microbe interactions, it is interesting that trehalose is regularly found in plant roots interacting with antagonistic fungi, mycorrhizal fungi, and in nitrogen-fixing root nodules, probably as a microbial substance. The impact of trehalose on plant metabolism and its role in nitrogen fixing symbiosis is unclear. This work focuses on the nodule symbiosis. It represents a genetic approach to study the role of trehalose synthesis by the microsymbiont. One pathway for trehalose synthesis is the OtsA/B pathway. Trehalose is synthesized from UDP-glucose and glucose-6-phosphate in a two-step process by the action of trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB). Homologues of the genes coding for these two enzymes in Escherichia coli, otsA and otsB, have been localized on the symbiotic plasmid of Rhizobium sp. NGR234 (pNGR234a). To study the significance of rhizobial trehalose synthesis in free living and symbiotic rhizobia, an Ω-cassette was inserted into the otsA homologue. Phenotypically, the deletion of the rhizobial otsA-homologue strongly reduced trehalose synthesis under microaerobic growth conditions. Thus, there are strong indications that the rhizobial trehalose synthesis induced under hypoxic conditions is directed by the symbiotic plasmid encoded otsA-homologue in conjunction with otsB. The functionality of otsA has therefore indirectly been demonstrated in Rhizobium sp. NGR234, which is the first time in aproteobacteria in general. In addition, the induction of otsA and its homologues by low oxygen conditions has not been previously reported. The natural environment inside nodules is characterized by low oxygen. In contrast, trehalose synthesis under salt stress was not influenced by the mutation of otsA. This indicates that Rhizobium sp. NGR234 exhibits a second trehalose pathway. Activities of maltooligosyltrehalose synthase and maltooligosyl trehalohydrolase (MOS – pathway) had been demonstrated in Rhizobium sp. NGR234 in previous work. To study the role of rhizobial otsA in symbiosis, various host plants were infected with Rhizobium sp. NGRWotsA. In a number of hosts, average nodule size was reduced, nodule number was increased (up to 30 %) and nitrogen fixation was reduced compared to control plants infected with the wildtype strain NGR234. Analysis of the carbohydrate content of these nodules revealed significant increases in the levels of sucrose, hexoses and starch. Thus the deletion of the potential rhizobial otsA-homologue has a severe impact on rhizobium-legume symbiosis, and a signal function of trehalose in carbohydrate partitioning and root nodule development is proposed.

There are some kinds of beneficial symbiotic and nonsymbiotic association between different soil microbes such as arbuscular mycorrhizal (AM) fungi and plant growth-promoting rhizobacteria (PGPR) with their host plants, resulting in the establishment of a sophisticated natural network. The growth of AM fungal spores results in the production of an extensive hyphal network, which can significantly increase the uptake of nutrients and water by the host plant. In the bacterial symbiosis, like rhizobium (as PGPR), the bacteria are able to initiate some cellular structures (nodules), which are actually plant-differentiated tissues and fix the atmospheric nitrogen (N) to be used by the host plant. For the initiation of such kind of symbioses and hence the establishment of the network, signal molecules must be exchanged between the two symbionts. Signal molecules are some kind of biochemical molecules, produced by plant roots and microbes, triggering genetic activation in both symbionts. However, there are some differences differentiating microbial symbiotic association from each other. For instance, AM fungal species are able to colonize a wide range of host plants, with their signal molecules indicating their nonspecific symbiotic association, while rhizobium bacteria are able to establish symbiosis with their specific host plant, which is due to the nature of their signal molecules. It is, therefore, important to indicate the precise details regarding the signal molecules including the plant hormones, which can establish such kind of symbioses and network and the interactions between the microbes. These details can be useful for the production of more efficient inoculums and a more productive and healthy environment. The most recent advancements are presented.

Fungal connections among plants, popularly known as the “wood wide web,” captured the interest of scientific and public audiences because these connections may facilitate increased growth, improved survival, nutrient transfer, and communication among plants. Research on these fungal networks has focused almost exclusively on known plant symbionts called mycorrhizal fungi. However, many non-mycorrhizal fungi also form ecologically important associations with plants. If non-mycorrhizal fungi such as Dark Septate Endophytes (DSEs) can form common networks among plants, then fungal connections among plants are likely more complex and prevalent than previously thought. In this study, we ask whether a common DSE can form hyphal connections between plants, improve plant biomass, and move water between them. Using a lab system with donor and receiver plants, we find that DSE hyphae crossed air gaps to physically connect plants. Receiver plants that were connected to a fungal network had higher biomass than those that were not. A water-soluble dye injected into donor plant leaves was detected in receiver leaves, but only when plants were connected via the fungal network. These results provide the first lab-based evidence that common non-mycorrhizal networks can occur and suggest that fungal networks among plants may extend beyond mycorrhizal fungi.

Root fungal endophytes are present in most plants and co-occur with other mycorrhizal fungi. Their intraradical colonization suggests a special, differentiated relationship with host plants and increases opportunities for close interactions between hosts and fungal symbionts (e.g., carbon to nutrient exchange or hormone signalling). During the symbiosis between plant and arbuscular mycorrhizal fungi (AMF), specific trading features are established. These features have been incorporated into the biological market hypothesis, where dynamics of carbon to nutrient trading in the plant‐mycorrhizal fungal mutualism are compared to trades in a market economy. Multiple examples of similar dynamics have been shown for root endophytic fungi: soil nutrients are transported to the plant in exchange for carbon. However, plants have been shown to be able to reward AMF that exchange larger amount of nutrients (and vice versa), while at least some root fungal endophytes have been described as “by-product mutualists”, where the fungal symbiont enhances the performance and fitness of their host plant by providing benefits, but not requiring major investments from the host. Whereas AMF have received large attention, the role of fungal endophytes in carbon to nutrient exchange with plants remains largely uninvestigated.In this study we aimed at developing a controlled system to evaluate effects of multiple fungal endophytes (Colletotrichum tofieldiae and Cladophialophora chaetospira) on a model plant species (Lotus japonicus) and their role in the carbon to nitrogen exchange in the presence of different nitrogen sources (organic and inorganic). We further developed this controlled system to include plants colonized by AMF. Two-compartment petri dishes were used to achieve plant root colonization in a nutrient limited compartment and allow separation of nutrient sources only accessible by the fungal endophytes. We performed dual 15N and 13CO2 pulse-labelling experiments to trace the fate of plant carbon into fungal biomass and of different nitrogen sources into plant aboveground tissues. We analysed root for RNA sequencing to gain insights into the genetic controls over the observed dynamics.We successfully established a controlled system and found that C. tofieldiae can elicit positive effects on plant growth and nitrogen acquisition. These effects are dependent on the nutrient source to which the fungus has access to, with positive effects displayed in the presence of organic nitrogen. Plants exchange relatively less carbon to C. tofieldiae accessing organic nitrogen. Root transcriptome shows specific changes in response to root fungal colonization which are dependent on the nitrogen source available to the fungal endophyte. Furthermore, the presence of AMF did not modify the observed carbon to nitrogen exchange dynamics.In conclusion we show that the root fungal endophyte C. tofieldiae can play an important role for plant nutrient acquisition in the presence of organic nitrogen. The trade of nitrogen for plant carbon displays different features from the AMF symbiosis (i.e., higher amount of nitrogen is not rewarded with plant carbon investment) and different gene regulations are involved. Our results indicate complementarity between C. tofieldiae and AMF during root colonization, offering mechanistic explanations for the concomitant presence of AMF and fungal endophytes in terrestrial ecosystems.

Summary From the phytocentric perspective, a mycorrhizal network (MN) is formed when the roots of two or more plants are colonized by the same fungal genet. MNs can be modelled as interaction networks with plants as nodes and fungal genets as links. The potential effects of MNs on facilitation or competition between plants are increasingly recognized, but their network topologies remain largely unknown. This information is needed to understand the ecological significance of MN functional traits. The objectives of this study were to describe the interaction network topologies of MNs formed between two ectomycorrhizal fungal species, Rhizopogon vesiculosus and R. vinicolor, and interior Douglas‐fir trees at the forest stand scale, identify factors leading to this structure and to contrast MN structures between forest plots with xeric versus mesic soil moisture regimes. Tuberculate mycorrhizas were sampled in six 10 × 10 m plots with either xeric or mesic soil moisture regimes. Microsatellite DNA markers were used to identify tree and fungal genotypes isolated from mycorrhizas and for comparison with reference tree boles above‐ground. In all six plots, trees and fungal genets were highly interconnected. Size asymmetries between different tree cohorts led to non‐random MN topologies, while differences in size and connectivity between Rhizopogon species‐specific subnetwork components contributed towards MN nestedness. Large mature trees acted as network hubs with a significantly higher node degree compared to smaller trees. MNs representing trees linked by R. vinicolor genets were mostly nested within larger, more highly connected R. vesiculosus‐linked MNs. Attributes of network nodes showed that hub trees were more important to MN topology on xeric than mesic sites, but the emergent structures of MNs were similar in the two soil moisture regimes. Synthesis. This study suggests MNs formed between interior Douglas‐fir trees and R. vesiculosus and R. vinicolor genets are resilient to the random loss of participants, and to soil water stress, but may be susceptible to the loss of large trees or fungal genets. Our results regarding the topology of MNs contribute to the understanding of forest stand dynamics and the resilience of forests to stress or disturbance.

<p>Mycorrhizal fungi are an important partner of almost all land plants, who trade soil nutrients, such as Phosphorus or Nitrogen, for photosynthetic Carbon (C). Moreover, mycorrhizal fungi connect multiple plants with their mycelium in so called Common Mycorrhizal Networks (CMNs). CMNs formed by ectomycorrhizal (EM) fungi are an inherent part of boreal and temperate forests, often termed the ‘wood-wide web’. However, the role of these networks for plant belowground C allocation and distribution is not well known.</p><p>Here, we examined how plant photosynthates are distributed within EM mycelium networks connecting pairs of young beech trees, addressing the following questions: (1) Is the total belowground C allocation of plant photosynthates influenced by the size of the mycorrhizal network and its access to resources? (2) Is the belowground C distribution within a CMN altered if trees have unequal access to C from photosynthesis? (3) Do CMNs amplify or alleviate competition for nutrients between connected trees?</p><p>We planted young beech trees in pots in a special two-plant box set-up which allows to control the establishment of mycorrhizal networks between them. For this, two plant pots, penetrable by fungal hyphae but not by roots, were placed inside of plastic boxes and the interstitial space was filled with quartz sand. In addition, a hyphal-exclusive N source consisting of <sup>15</sup>N labeled peat (‘peat bag’), was buried within each plant pot. Two treatments were applied in a fully factorial design: 1) Allowing/preventing the establishment of a CMN between the pots (some pots were turned around at a regular interval to prevent the establishment of CMNs) and 2) inequality of access to photoassimilated C (in part of the boxes one of the two plants was shaded). In a <sup>13</sup>C-CO<sub>2</sub> labeling approach, we traced <sup>13</sup>C assimilated by one plant of each tree pair into belowground pools of both plants by isotope ratio mass spectrometry (EA-IRMS) and <sup>13</sup>C phospholipid fatty acid (PLFAs) analysis (GC-IRMS). At the same time, we investigated plant uptake of <sup>15</sup>N via mycorrhiza by EA-IRMS.</p><p>Our results demonstrate that plants relied mostly on their fungal partners to acquire nutrients (63% of plant N was derived from mycorrhiza-exclusive peat bags), and also directed the majority of the C allocated belowground to their mycorrhizal partners. The presence of a larger mycorrhizal network connecting to another plant and an additional N source almost doubled photosynthetic CO<sub>2</sub> assimilation and belowground C allocation by plants. Fungi translocated carbon via hyphal linkages preferentially into mycorrhiza-exclusive nutrient patches, even when they were located within the realm of a neighboring plant and this necessitates to cross a nutrient-poor zone of sand. Shading did not affect the belowground distribution of C.</p><p>We conclude that belowground ectomycorrhizal networks represent a significant sink strength for plant photosynthates and may thus be a major driver of C sequestration in beech forest soils. The belowground distribution of C via fungal networks is mainly related to the distribution of nutrient-rich patches in the soil and less to differences in the photosynthetic capacity of the host plants.</p>