Soil microbiomes and neighbouring plants both greatly influence plant growth, thereby shaping community dynamics. Although these processes have been studied extensively, they are rarely integrated in a mechanistic framework. Consequently, how they interact mechanistically remains poorly understood. Here we address these questions using a plant-soil feedback approach with six grassland species grown alone or with five different neighbours in sterile soil and in soils with legacies of conspecific and heterospecific plants. Focal plants experienced stronger growth suppression by neighbours in sterile soil, but this was alleviated by microorganisms in soils with legacies, and particularly in soils with heterospecific legacies. Plants consequently often performed better with a heterospecific neighbour than with a conspecific one and in many cases reached equal or even bigger size than when growing alone. Neighbours restructured the root-associated bacterial community of the focal plant towards that of the neighbour, with stronger convergence in neighbour legacy soils, and these shifts mirrored the growth responses of the focal plants. Importantly, the degree of this microbial convergence predicted growth responses. An independent inoculation experiment with natural bacterial communities cultured from focal plants supported the idea that neighbour-induced bacterial community shifts contribute to these growth responses. Our results reveal that neighbour-induced bacterial reassembly, modulated by soil microbial legacies, mediates plant-plant interactions. This mechanism provides new insights into how soil microbiomes can integrate with direct plant interactions, which can ultimately influence coexistence and competition in grassland ecosystems.

Root-associated microbiomes are widely assumed to be primarily shaped by the host plant. However, other co-occurring plants can also influence the composition of this microbiome. This can be either directly, by stimulating or suppressing microbial recruitment of the focal plant, or indirectly, through soil legacies that shape the microbial pool encountered by the focal plant during establishment. The relative importance of these neighbor effects shaping the focal plant microbiome and the consequences for focal plant growth remain poorly known. Here, we challenge the paradigm that the host plant identity is the main driver of root-associated bacterial and fungal communities. Under experimental conditions, we show that the influence of the host plant on root-associated microbiomes is dramatically reduced in the presence of neighbors and their soil legacies. Remarkably, root-associated bacterial communities were primarily shaped by co-occurring neighbor plants, while fungal communities were mainly determined by soil legacies from previously grown plants. Furthermore, culturable microbial communities (NatComs) isolated from the roots of focal plants differed greatly depending on neighbor identity and soil legacies. Inoculating new focal plants with these communities caused strong responses in growth that varied greatly depending on the history of these isolated communities, highlighting the importance of neighbor effects. Finally, we show that metabolites produced by these microbial communities are one mechanistic pathway through which neighbors influence plant growth. Our results stress the often-overlooked ecological and functional importance of both direct and indirect effects of neighbor plants in shaping root-associated microbiomes. By integrating neighbor effects with host identity, this work provides a starting point for understanding how neighboring plants contribute to shaping plant communities, with potential implications for sustainable agriculture.

Growing knowledge on plant microbiomes demonstrates the contribution of the host plant during microbiome assembly, especially under stress conditions commonly threatening crops. To dissect the influence of a plant on its microbiome, repeated cycling of microbiomes can be utilized to enhance functional properties in the enriched microbial communities. We used such a successive cultivation approach for wheat (Triticum aestivum) microbiome under drought conditions and selected lineages for drought resilience and susceptibility, with and without enriching the starting community with a library of bacterial isolates obtained from wheat. Significant differences in the rhizosphere microbiome between selection regimes were confirmed through 16S rRNA gene amplicon sequencing. Notably, replicate lineages of each selection regime showed convergence to similar microbiomes. Specific genera were abundant depending on the selection regimes; Stenotrophomonas under drought resilience, while Rahnella under drought conditions when the strain library was added initially. Applying Stenotrophomonas or Rahnella as single inoculum did not improve drought resilience in wheat. We hypothesize that complex microbiome dynamics take place during successive cultivation, which underscores the importance of considering complex plant-microbiome systems for studying plant stress resilience. Successive cultivation remains a valuable approach for observing rhizosphere microbiome changes under different conditions.

Biodiversity is known to influence disease risk, yet the pathways of pathogen transmission within plant communities remain poorly understood, especially belowground. In particular, how soil-borne pathogens move from resident vegetation and soil to colonize new hosts is unresolved. We traced belowground pathogen transmission using phytometer seedlings of two plant species planted in a long-term grassland biodiversity experiment. After 3 months, we characterized the fungal communities of phytometer roots, resident plant roots, and soil using high-throughput sequencing and the FungalTraits database to identify associations between pathogen taxonomy and plant families. Next, we related pathogen abundance to phytometer growth. The phylogenetic similarity of phytometers with resident plant species strongly predicted the relative abundance of pathogens that were considered family-specific, but not of pathogens without a clear host preference. However, neither pathogen abundance in phytometers nor resident plant biomass affected phytometer growth, which was best explained by the resident communities' species richness. Combining sequencing of fungal communities with in situ field manipulations enabled us to track the associations between multiple soil-borne pathogens and plant hosts within the full complexity of plant-soil systems. While pathogen dynamics were readily detectable, their consequences for plant performance may only become apparent over longer ecological timescales.

Mycorrhizal symbioses are prevalent in terrestrial ecosystems and play essential roles in plant nutrition and health. However, the relative importance of plant evolutionary history, physiology, and eco-geographical factors in shaping mycorrhizal fungal community assembly remains poorly understood. Here, we investigate how plant phylogeny, trophic mode, biogeographic distribution and environmental niche collectively influence the diversity and composition of mycorrhizal fungal communities across the Orchidaceae, spanning broad phylogenetic and ecological scales. By using family-wide orchid-fungal associations and global occurrence data, our analyses showed that the variation in fungal diversity and community structure can be partially explained by orchids’ trophic mode, biogeographic distribution and environmental niche, but not by their overall phylogenetic relatedness. Among trophic modes, partially mycoheterotrophic orchids exhibited the highest level of fungal diversity (the lowest level of fungal specificity) in association with a broad range of phylogenetically dispersed fungal partners. Between biogeographical regions, a significantly higher level of fungal specificity was found for orchid species distributed in Australia than those in Eurasia and Africa. Furthermore, multivariate analyses showed that a small portion of the variation in fungal community structure was significantly related to broad climate, soil and vegetation variables, indicating the existence of large-scale habitat filtering on orchid mycorrhizal communities. Altogether, our findings indicate that mycorrhizal communities in the orchid family are likely shaped by multiple, intertwined factors related to orchid ecophysiology and biogeography on a global scale.

Afforestation is increasingly recognized as a critical strategy to restore ecosystems and enhance biodiversity on post-agricultural landscapes. However, agricultural legacies, such as altered soil structure, nutrient imbalances, and depleted microbial diversity, can slow down forest establishment or cause ecosystems to deviate from expected successional trajectories. In this opinion paper, we explore the potential of soil inoculations as a tool to overcome these challenges by introducing beneficial microbial communities that can accelerate ecosystem recovery and forest development. Restoring soil biodiversity is a crucial aspect of this process that drives broader ecosystem functionality and resilience. We highlight the need to carefully consider the type and timing of inoculations and to ensure compatibility between the inoculum and recipient site characteristics to optimize the establishment of introduced species. While tree productivity is often a central focus of afforestation efforts, the restoration of soil biodiversity, which will also contribute to increased ecosystem-level functions, should also be a priority for long-term forest resilience. Agricultural legacies add complexities to the restoration process, creating unique challenges that need to be addressed in restoration planning. Thus, successful inoculation strategies require a thorough understanding of both donor and recipient site characteristics, also in relation to potential mismatches related to soil physiochemical properties to avoid unintended consequences such as the non-establishment of introduced species. Additionally, we call for the re-evaluation of afforestation targets and the development of standardized monitoring protocols that track the success of inoculation efforts, particularly regarding soil health, microbial community establishment, and biodiversity recovery. By integrating inoculation practices within a broader restoration framework, we can enhance the resilience, biodiversity, and ecosystem functionality of newly afforested landscapes. Ultimately, this approach may play a critical role in ensuring the success of large-scale afforestation projects.

Integration of soil biodiversity restoration into reforestation strategies is important to accelerate the restoration process given the interplay between belowground and plant community dynamics. Research on the mechanisms underlying the temporal changes that occur in soil communities has been limited, and this is especially true for eukaryotes. Understanding these processes can help us to manipulate soil communities in a way that promotes recovery and stability. Here, we test the importance of selection, dispersal, and drift in structuring community composition of taxonomic and functional groups of soil eukaryotes in croplands and planted forests, and their relationships with successional time. We observed a link between functional/taxonomic groups of soil eukaryotes, the importance of ecological processes in their community compositional patterns, and successional time. To optimize the effectiveness of soil restoration, we suggest implementing additional measures that target specific groups of soil organisms, account for the key ecological processes structuring their communities, and address how these processes correlate with successional time. Our findings advance both theoretical understanding and practical guidelines for restoring soil biodiversity.

Trees are essential to ecosystems in both natural and urban environments, yet they are increasingly threatened by abiotic and biotic stresses linked to climate change and human activities. The use of microbial-based approaches and microbiome engineering to safeguard plants and crop production is promising, but their application in trees raises specific challenges. Here, we review knowledge on the tree microbiome and outline opportunities to leverage tree-associated microbial communities. We describe the specific challenges inherent to working with tree species and highlight how Synthetic Microbial Communities (SynComs) can be used to study and engineer tree microbiomes. Finally, we propose that future research priorities include (1) developing model tree systems for experimental work, (2) obtaining tree-specific culture collections and SynComs, and (3) optimising methods for tree SynCom inoculation. Unlocking these methodological challenges will enable us to realise the potential of the tree microbiome and address global challenges in tree health and the provision of ecosystem services.

Nitrogen (N) is essential for plant growth, yet excessive fertilizer use contributes to environmental degradation. Actinorhizal trees like Alnus glutinosa form symbiotic relationships with nitrogen-fixing bacteria of the genus Frankia, reducing reliance on synthetic fertilizers. However, distinguishing between soil-derived and symbiotically fixed nitrogen remains a challenge. This study investigates the potential of NIR spectroscopy as a nondestructive tool for differentiating N sources in A. glutinosa. Seedlings were grown in sterilized soil under controlled conditions with and without Frankia inoculation, and across a gradient of NH4NO3 fertilization (0-20 mM). We measured leaf chlorophyll, nitrogen content, biomass, and NIR reflectance (330-1100 nm) of the third fully expanded leaf. principal component analysis (PCA) and partial least squares (PLS) regression revealed that spectral signatures significantly differed between inoculated and uninoculated plants, particularly in the visible range around 555 nm. Despite similar leaf chlorophyll levels, Frankia-inoculated plants and those fertilized with 20 mM NH4NO3 exhibited spectral differences that could otherwise not be detected by SPAD measurements. PLS regression explained up to 54.8% of spectral variance based on nitrogen source, even in the absence of unique spectral peaks. These findings highlight the potential of NIR spectroscopy for rapid, in vivo and in vitro assessment of symbiotic N-fixation in trees, offering a novel and more precise approach than SPAD measurements.

The symbiotic relationship between the nitrogen-fixing bacteria Frankia alni and the pioneer tree species Alnus glutinosa plays an important role for tree performance, helping trees thrive in nitrogen-poor environments. However, F. alni nodulation can vary greatly between different soils and this could be due to biotic and abiotic characteristics of the soil. Here we examine how microbial communities from young (similar to 10 years) and mature (>= 100 years) forests and gradients of available nitrogen (N) and phosphorus (P) influence F. alni nodulation and tree performance. In mesocosm experiments, A. glutinosa seedlings were inoculated with bacteria, fungi, or both, cultured from young and mature forest soils, alongside F. alni. The impacts of N and P availability were examined through controlled nutrient manipulations. Results demonstrated that fungal communities from mature forests suppressed the growth-promoting effects of F. alni, although nodule biomass itself was not directly influenced. Further, we isolated and identified bacteria and fungi that were found to contribute to the observed inhibitory effects on F. alni-mediated growth promotion. Increased N availability significantly reduced nodule biomass, and we established a threshold at which reliance on symbiosis diminished. Conversely, P-addition stimulated nodulation and tree growth. These findings highlight the pivotal influence of N and P availability in determining the A. glutinosa-F. alni symbiosis, while also providing evidence that specific microbes in the soil influence these dynamics.

Soil communities are essential to ecosystem functioning, yet the impact of reducing soil biota on root-associated communities, tree performance, and greenhouse gas (GHG) fluxes remains unclear. This study examines how different size fractions of soil biota from young and mature forests influence Alnus glutinosa performance, rootassociated community composition, and GHG fluxes. We conducted a mesocosm experiment using soil community fractions (wet sieving through 250, 20, 11, and 3 mu m) from young and mature forest developmental stages as inocula. The results indicate that the root-associated community composition was shaped by forest developmental stage but not by the size of the community fractions. Inoculation with the largest size fraction from mature forests negatively affected tree growth, likely due to increased competition between the plants and soil biota. In addition, GHG fluxes were not significantly impacted by either size fraction or forest developmental stage despite the different community composition supplied. Overall, our research indicates that A. glutinosa strongly selects the composition of the root-associated community, despite differences in the initial inoculum, and this composition varies depending on the stage of ecosystem development, impacting the performance of the trees but not GHG fluxes.

Societal Impact StatementTrees in cities provide a great number of benefits to people and nature, but they are challenged by harsh conditions. Trees rely on helpful fungi in their roots to get essential nutrients from the soil, but we do not know which of these fungi are resistant to city landscapes. By studying these fungi, we can learn how their communities are affected by cities and which of them survive best there. Understanding this can help us develop effective ways to make use of these microbes or to improve city conditions for the benefit of urban trees and their fungi and, therefore, us.Summary Urban trees are important green features in cities. Arbuscular mycorrhizal fungi (AMF) in roots may alleviate urban environmental pressures affecting urban trees. These pressures also appear detrimental for AMF, reducing root colonization rates, spore production and spore diversity. However, a limited number of molecular studies on urban AMF contrast these findings, but the matter remains unresolved. Therefore, we investigated the hitherto understudied AMF communities in the roots of urban trees, their diversity, spatial and neighbouring‐tree effects and interaction networks. We did this by metabarcoding fungi from tree roots across an urbanization gradient in Amsterdam (the Netherlands), focussing on Dutch elm (Ulmus x hollandica). Samples were collected from three urbanization classes, differentiated by the degree of soil sealing and management: an urban forest, park and street. In the urban forest and park, root samples were collected in a grid, allowing the construction of interaction networks and assessment of neighbouring‐tree root effects, whereas the street trees stood in solitary pits. Using the ITS2 barcode, we detect distinct, diverse and heterogenous AMF communities. The (phylogenetic) diversity of AMF increased with urbanization. Contrary to our expectations, we found no evidence that AMF communities in street trees were more spatially homogenous. Moreover, neighbouring root diversity did not appear to affect AMF diversity. Our findings suggest a strong response of AMF communities to urbanization. An urban‐induced change in mycorrhizal partners, rather than a loss of partners and interaction complexity, demonstrates the high adaptability of the arbuscular mycorrhizal symbiosis to urban stressors, which has important management implications.

The prevalence and potential functions of common mycorrhizal networks, or the ‘wood-wide web’, resulting from the simultaneous interaction of mycorrhizal fungi and roots of different neighbouring plants have been increasingly capturing the interest of science and society, sometimes leading to hyperbole and misinterpretation. Several recent reviews conclude that popular claims regarding the widespread nature of these networks in forests and their role in the transfer of resources and information between plants lack evidence. Here we argue that mycoheterotrophic plants associated with ectomycorrhizal or arbuscular mycorrhizal fungi require resource transfer through common mycorrhizal networks and thus are natural evidence for the occurrence and function of these networks, offering a largely overlooked window into this methodologically challenging underground phenomenon. The wide evolutionary and geographic distribution of mycoheterotrophs and their interactions with a broad phylogenetic range of mycorrhizal fungi indicate that common mycorrhizal networks are prevalent, particularly in forests, and result in net carbon transfer among diverse plants through shared mycorrhizal fungi. On the basis of the available scientific evidence, we propose a continuum of carbon transfer options within common mycorrhizal networks, and we discuss how knowledge on the biology of mycoheterotrophic plants can be instrumental for the study of mycorrhizal-mediated transfers between plants. In this Perspective, Vincent Merckx and colleagues discuss an important but overlooked aspect of mycorrhizal interactions, mycoheterotrophy, in the context of recent arguments about the importance of these interactions to forest functioning.

During early afforestation stages, biotic and abiotic soil characteristics change at different paces. However, the extent that each of these characteristics contribute to plant performance and subsequent herbivory remains unclear. This study aimed to study the effects of biotic and abiotic characteristics of forest soil on Alnus glutinosa performance and their subsequent impact on foliar herbivory during early afforestation. Soils were collected from a series of replicated forests of 10, 15 or 25 years old, planted in agricultural soils. Two experiments were conducted, focusing on the effect of soil microbiome (live vs. sterilized forest soil, and bulk sterilized soil vs. bulk inoculated with forest soil) and forest age on tree performance, root-associated microbial communities, and plant-herbivore interactions. In 10-year-old forest soil, A. glutinosa stems were thinner when grown in sterilized soil than when grown in live soil. In 15- and 25-year-old soil, trees exhibited lower fine root percentages and thicker stems in sterilized than in live soils, suggesting age-dependent responses possibly arising from plant and microbe nutrient competition. Overall root-associated microbial communities showed no significant differences in their composition based on forest ages. Streptomyces sp. and Rokubacteriales were differentially more abundant in the roots of trees growing in 15-year-old soils and their relative abundance was correlated positively with aboveground biomass, suggesting that effects of forest age on tree performance are contingent on the unique microbiome of each forest. The herbivory assay using Mamestra brassicae larvae revealed a positive correlation between leaf nitrogen content and leaf area consumption in the live vs. sterilized soil experiment, but not in the inoculated soil experiment. Trees in 10-year-old forest soils exhibited the highest herbivore performance, suggesting heightened herbivore susceptibility in early afforestation. Our findings underscore that intricate relationships between soil conditions, microbial communities, and plant-herbivore interactions affect tree performance and herbivory during early afforestation.

Aims The spread of invasive weeds threatens biodiversity and stability of ecosystems. Jacobaea vulgaris is an invasive weed in some countries and an outbreak species in its native European range. Although biological control using specialist herbivores is available, controlling with soil microorganisms remains far less explored.Methods Twenty bacteria strains isolated from roots of J. vulgaris were used to examine bacterial effects on seed germination, root morphology and early plant growth. Moreover, we tested direct effects of the bacteria on a specialist herbivore of J. vulgaris, the leaf chewing caterpillar (Tyria jacobaeae), commonly used in biocontrol. We also tested indirect effects of bacteria, via the plant, on the performance of T. jacobaeae and the aphid species Aphis jacobaeae. Lastly, we examined the host specificity of two tested bacteria on three other forbs.Results Two Gammaproteobacteria, Pseudomonas brassicacearum and Serratia plymuthica, significantly reduced root growth of seedlings in-vitro, while seed germination was unaffected. However, these negative effects were observed across other forb species as well. Bacillus spp. injection led to the highest T. jacobaeae caterpillar mortality, while ingestion had no effect. Inoculation of the plants with bacteria did not affect aphid performance, but significantly affected T. jacobaeae preference. Specifically, P. syringae and one Bacillus sp. strain significantly increased T. jacobaeae preference.Conclusions Our results show that two root-associated bacteria inhibit J. vulgaris growth, but their lack of host specificity restricts their potential for biocontrol. Our study also highlights that belowground microorganisms can hamper or enhance the performance of aboveground insects.

Meadows and forests are the main vegetation types in temperate terrestrial ecosystems, and largely contribute to soil carbon (C) stock. Bioavailable C inputs can accelerate microbial decomposition of soil organic matter (SOM), which is known as "priming effect". However, it is still unclear how priming effect, as an important mechanism influencing soil C sequestration, is influenced by spatial transition of vegetation from meadow to forest. To investigate the mechanism of priming effect along a spatial transition gradient of vegetation, a soil incubation experiment with 14C labeled glucose was combined with microbial rDNA sequencing and gene composition prediction. The results showed that with the vegetation transition from meadow to forest soil available phosphorus (P) significantly increased, in contrast to dissolved nitrogen (N) and C which remained unaffected. Moreover, the soil microbial community composition shifted towards a higher relative abundance of K-strategists (Acidobacteria) and a lower abundance of r-strategists (Actinobacteriota) along the vegetation transition from meadow to forest. In the meadow, the microbial community consumed more of the added glucose and increased priming effect. This was accompanied by lower available P but higher soil bacterial gene function encoding P cycling. In contrast, increased soil P availability in forest soils caused a decelerated microbial metabolism of phosphorylated organic compounds within microbial biomass due to decreased microbial demand for P acquisition from SOM, and thus resulted in suppression of the priming effect. Our study showed that P availability and microbial community shifts in spatial transition zones between meadows and forests are important drivers for the priming effect on SOM decomposition.

Stable isotope signatures of fungal sporocarps have been instrumental in identifying carbon gains of chlorophyllous orchids from a fungal source. Yet, not all mycorrhizal fungi produce macroscopic sporocarps and frequently fungi of different taxa occur in parallel in orchid roots. To overcome this obstacle, we investigated stable isotope signatures of fungal pelotons extracted from orchid roots and compared these data to the respective orchid and reference plant tissues. Anoectochilus sandvicensis and Epipactis palustris represented specialized or unspecialized rhizoctonia-associated orchids. Epipactis atrorubens and Epipactis leptochila are orchids considered ectomycorrhiza-associated with different preferences for Basidio- and Ascomycota. 13 C enrichment of rhizoctonia pelotons was minor compared with plant tissues and significantly lower than enrichments of pelotons from ectomycorrhizal Epipactis species. 15 N values of pelotons from E. leptochila and E. atrorubens showed similar patterns as known for respective sporocarps of ectomycorrhizal Ascomycota and Basidiomycota, however, with an offset towards lower 15 N enrichments and nitrogen concentrations. Our results suggest an explicit fungal nutrition source of orchids associated with ectomycorrhizal fungi, whereas the low 13 C enrichment in rhizoctonia-associated orchids and fungal pelotons hamper the detection of carbon gains from fungal partners. 15 N isotopic pattern of orchids further suggests a selective transfer of 15 N-enriched protein-nitrogen into orchids.

Since the first discovery of unique carbon (C) and nitrogen (N) isotope signatures in fungal fruiting bodies (Gebauer & Dietrich, 1993; Gleixner et al., 1993), natural abundances of stable isotopes have been extensively used to identify the nutritional dynamics of fungi (Mayor et al., 2009). Assigning ecological roles of fungi is essential to determine the role of individual taxa in nutrient cycling and forest ecology. The use of isotope natural abundances in forest ecosystems has been crucial in distinguishing fungi with two main modes of life: ectomycorrhizal and saprotrophic fungi (Henn & Chapela, 2001). Within saprotrophic fungi, isotope natural abundances further allow the identification of the substrates used (Kohzu et al., 1999). Dual isotope analyses of the δ13C and δ15N values consistently indicate a differentiation in isotopic signatures between ectomycorrhizal and saprotrophic fungi within and among ecosystems (Henn & Chapela, 2001; Taylor et al., 2003; Trudell et al., 2004; Mayor et al., 2009). These signatures have been shown to reflect the ecophysiology of fungi and demonstrate that fungi that can utilize organic nitrogen exhibit higher δ15N than those fungi restricted to mineral nitrogen sources (Gebauer & Taylor, 1999; Lilleskov et al., 2002). Still, the ability to distinguish fungal nutritional modes has been long restricted to fungi that produce macroscopic sporocarps, such as mushrooms, due to their large mass which allows for physical measurements. Thus, for many fungi, particularly those associated with plant roots that do not form evident fruiting bodies, isotope natural abundances of fungal hyphae are scarce. Besides ectomycorrhizal fungi, isotope natural abundances are known for sporocarp-forming ericoid (e.g. Hobbie & Hogberg, 2012) and orchid-associated nonrhizoctonia saprotrophic fungi (e.g. Ogura-Tsujita et al., 2009). Yet, values of δ13C and δ15N are poorly known for arbuscular mycorrhizal fungi (but see e.g. Courty et al., 2011; Suetsugu et al., 2020, for isotope values of fungal spores), and the orchid-associated fungi known as ‘rhizoctonia’ in natural conditions. Recently, Klink et al. (2020) obtained the δ13C and δ15N of arbuscular mycorrhizal hyphae isolated from roots of a grass and a legume, inoculated in experimental conditions, thereby providing an efficient method to extract hyphae from roots. Using this method with a few modifications, here, we measured the isotope natural abundances δ13C and δ15N of naturally occurring arbuscular mycorrhizal (Fig. 1a–c) and orchid-associated hyphae (Fig. 1d–f) directly from roots (see Supporting Information Methods S1). To obtain hyphae of arbuscular mycorrhizal fungi, we selected two species of fully mycoheterotrophic plants: Thismia megalongensis C. A. Hunt, G. Steenbee. & V. Merckx and Sciaphila megastyla Fukuy. & T. Suzuki. Mycoheterotrophs are achlorophyllous plants that obtain carbon from their associated fungal partners (Leake, 1994; Merckx, 2013). Species in the plant genus Thismia have been demonstrated to be highly specialized on narrow lineages of Glomeromycotina fungi (Gomes et al., 2017; Merckx et al., 2017), while species of Sciaphila tend to associate with a wider phylogenetic diversity within the fungal subphylum (Merckx et al., 2012; Suetsugu & Okada, 2021). For fungi associated with orchid roots, we selected two chlorophyllous partially mycoheterotrophic orchid species, known to associate with rhizoctonia symbionts, Orchis militaris L. and Ophrys insectifera L., for which both isotope natural abundances and Sanger sequencing of the root-associated fungi have been performed previously (Schweiger et al., 2018). To be able to compare isotope values across sampling sites, the δ values of C and N stable isotope abundances were normalized by calculating enrichment factors (ε; see Methods S1). The enrichment factors ε13C and ε15N were significantly different between the fungal hyphae, mycoheterotrophic and reference plants for both T. megalongensis and S. megastyla (Fig. 1g; Table 1). For both species, ε13C was not distinguishable between the mycoheterotrophs and respective fungal hyphae, while ε15N was significantly different between mycoheterotrophs and fungi for S. megastyla, and marginally significant for T. megalongensis (Fig. 1; Table 1). In relation to the reference plants, the fungi extracted from both mycoheterotrophic species were significantly enriched in ε13C, and fungi from S. megastyla were marginally significantly depleted in ε15N. Similarly, both mycoheterotrophic plants were enriched in ε13C although only significantly for T. megalongensis. This indicates that the ε13C of fungal hyphae drives the 13C enrichment of arbuscular mycorrhizal fully mycoheterotrophic plants, and there seems to be a difference in nitrogen source between T. megalongensis and S. megastyla-associated fungi. Each mycoheterotrophic plant species is associated with nonoverlapping fungal clades within the Glomeromycotina (Fig. 2a). Sciaphila megastyla harboured fungi belonging to the genera Dominikia, Kamienskia and two unidentified amplicon sequence variants, while the fungi in the roots of T. megalongensis belonged exclusively to the genus Rhizophagus, supporting a specialization on fungal interactions of different degrees between these plant lineages (Gomes et al., 2020; Suetsugu & Okada, 2021). The enrichment factors ε13C and ε15N were generally significantly different between fungal hyphae, orchids and reference plants for both O. militaris and O. insectifera (Fig. 1h; Table 1). Both ε13C and ε15N were significantly different between orchid leaves and hyphae for O. militaris, while for O. insectifera, only ε13C was significantly higher in the hyphae in comparison with the plant tissue (Fig. 1; Table 1). In both orchid species, fungal hyphae were significantly enriched in ε13C, and in O. insectifera fungi were also enriched in ε15N in relation to the reference plants. The fungal hyphae extracted from the two orchid species were only weakly enriched in ε13C in comparison with reference plants and far less enriched in 13C than tissues of ectomycorrhizal fungi reported previously (Mayor et al., 2009). This observation is consistent with previous findings of absence of 13C enrichment in fully mycoheterotrophic protocorms of O. militaris, which were also associated with rhizoctonia fungi by Schweiger et al. (2018). Interestingly, in that study, protocorms of O. insectifera were somewhat enriched in 13C. We detected most sequenced reads obtained from root pieces to belong to the fungal order Helotiales. Fungi in the genus Ilyonectria were also detected, concordant with previous observations of these orchid species collected at the same site (Schweiger et al., 2018). Both Helotiales and Ilyonectria were present in the roots of both orchid species and, as far as we know, have an unknown ecological function. Helotiales have also been detected in the species studied in Zahn et al. (2023). In addition, we detected rhizoctonia fungi belonging to the families Ceratobasidiaceae, Serendipitaceae and Thelephoraceae in the roots of O. insectifera, and to the families Ceratobasidiaceae and Thelephoraceae in the roots of O. militaris (Fig. 2b). One orchid individual of O. militaris presented a high relative abundance of Ceratobasidiaceae, and another of Thelephoraceae in their roots. We cannot exclude that Tulasnellaceae are underrepresented in our data influenced by the primers used (Vogt-Schilb et al., 2020), as these taxa have been shown to be present in O. insectifera roots (Schweiger et al., 2019). Besides rhizoctonia fungi, we also found fungi known to form ectomycorrhizas (according to FungalTraits; Põlme et al., 2020), such as Sebacina (Sebacinaceae), Amphinema (Atheliaceae), Hebeloma and Hymenogaster (Hymenogastraceae) in two O. insectifera individuals. In terms of isotope signatures, no apparent differences were observed between individual samples where rhizoctonia fungi are present and those where Helotiales are predominant, and neither in relation to the plant material between specimens. Yet, a larger sample size would be needed to properly evaluate this. While ε13C values of hyphae are within the same range as found for the respective plant tissues of the AM mycoheterotrophic plants, as expected, ε15N values of the hyphae were considerably lower than ε15N of the respective plant tissues. This relative 15N depletion of hyphae in comparison with plant tissue was also observed for the two orchid species. One could wonder whether this depletion is either due to potential loss of hyphal content during extraction considering that nitrogen in chitin is depleted in 15N by c. 10‰ in comparison with fungal protein (Taylor et al., 1997; Hobbie & Hogberg, 2012) or due to a selective transport of 15N-enriched protein-derived compounds from fungal to plant tissues. Similarly, Zahn et al. (2023) show an equal depletion in 15N of hyphae extracted from two rhizoctonia-associated orchid species and for identically extracted hyphae from ectomycorrhiza-associated orchid roots an even larger depletion in 15N in relation to orchid leaves. In addition, the N concentrations of the extracted fungal hyphae of both T. megalongensis (2.25 ± 0.53 mmol gdw−1) and S. megastyla (2.18 ± 0.42 mmol gdw−1) were not distinguishable from those of the mycoheterotrophic plant tissues (1.95 ± 0.28 and 1.88 ± 0.45 mmol gdw−1 respectively), in congruence with Klink et al. (2020), while reference plants presented lower N concentrations (1.36 ± 0.44 and 1.17 ± 0.19 mmol gdw−1 for each set respectively) in relation to both fungal hyphae (T. megalongensis: Z = 2.772, P = 0.008, and S. megastyla: Z = 3.231, P = 0.002) and mycoheterotrophic plants (T. megalongensis: Z = 2.140, P = 0.032 and S. megastyla: Z = 2.710, P = 0.007). The N concentrations between fungal hyphae (1.19 ± 0.23 mmol gdw−1 for O. militaris and 1.64 ± 0.17 mmol gdw−1 for O. insectifera), orchids (1.74 ± 0.07 and 2.19 ± 0.17 mmol gdw−1 respectively) and reference plants (1.62 ± 0.76 and 1.79 ± 0.79 mmol gdw−1 for each set respectively) were not statistically different for both orchid species. In Zahn et al. (2023), the fungal hyphae extracted from rhizoctonia-associated orchids were also nondistinguishable from reference plants, while for one species (Anoectochilus sandvicensis), fungal hyphae had significantly lower N concentration than the orchid leaves. The arbuscular mycorrhizal diversity in the roots of the mycoheterotrophic plant species did not overlap between T. megalongensis and S. megastyla, and the fungal enrichment in ε15N was variable between plant species. The association with different fungal genera, in addition to local soil nitrogen availability, could have contributed to the differences in ε15N between species. Further studies are required to assess the source of variation and generality of isotope values among arbuscular mycorrhizal fungi. In the orchid-associated fungi, the fungal composition was variable between individual specimens, yet without reflection on the isotopic values of the extracted hyphae. The absence of differences in fungal isotopic values may indicate an artefact on the integration of both techniques. The apparent dominance of specific fungal groups in the roots could reflect spatial segregation of fungi, as a small piece of root was used for sequencing, while for the hyphal extraction, the remainder of the root system was used. Furthermore, ectomycorrhizal fungi were detected in two individuals of O. insectifera, while rhizoctonia fungi were detected in three individuals. The presence of ectomycorrhizal fungi in the roots of some rhizoctonia-associated orchids is commonly reported in the literature (e.g. Jacquemyn et al., 2021), yet it remains to be demonstrated whether these fungi indeed establish a mycorrhizal symbiosis with the orchid, in a sporadic or constant way during the orchid development, or represent endophytic fungi as it has been shown in typical nonmycorrhizal hosts (Schneider-Maunoury et al., 2020). Nevertheless, we cannot exclude those ectomycorrhizal fungi found in the roots of O. insectifera to be responsible for the slight enrichment in 13C and 15N of the hyphae extracted from O. insectifera in comparison with O. militaris. However, these isotopic differences are rather small and are not seen in the leaves of these two species, that is there appears to be no major plant matter gain from these ectomycorrhizal fungi. In addition, our results reveal that sporadic appearance of ectomycorrhizal fungi in orchids hitherto classified as rhizoctonia-associated does obviously not affect their isotope signature. Zahn et al. (2023) present further isotope signatures and diversity of root-associated fungi of orchids associated with ectomycorrhizal fungi. The assessment of fungal diversity often comprises a qualitative snapshot of a fraction of the root system, and although different fungal species or guilds may contribute differently to nutrient uptake at multiple occasions, we still lack a solid framework to quantify the contribution of each of these fungi to fungal–plant matter exchange. By contrast, isotopic abundance data are a temporal and spatial integrator (Dawson et al., 2002) over all fungal–plant matter exchange processes without providing direct information about the role of the individual potential fungal players, which is less sensitive to occasional changes in carbon or nitrogen supply. To the best of our knowledge, we reveal for the first-time isotope signatures of hyphae of arbuscular mycorrhizal fungi in mycoheterotrophic plants and, together with Zahn et al. (2023), of fungal pelotons present in chlorophyllous orchids in relation to the plant tissues from their roots. Arbuscular mycorrhizal hyphae have isotope signatures that allow a significant distinction in ε13C abundance in relation to reference plants. Subsequently, hyphae resemble the ε13C of the mycoheterotrophic plants, suggesting that these mycoheterotrophs gain carbon from the detected fungi. For the orchid-associated fungi, hyphae are only slightly enriched in 13C in relation to both reference and orchid plants, remaining unclear whether these orchids gain C from the associated fungi based on these results. However, the significant enrichment in 15N of hyphae or orchid leaves indirectly indicates a partial mycoheterotrophic matter gain by these orchids. Our study appeals to a careful interpretation when integrating root-associated fungal diversity and isotope natural abundances considering their inherent ecological significance as each method contains fundamentally different categories of information. Still, the combination of both approaches is greatly valuable and contributes to understand complex patterns in plant–fungal interactions, for example considering spatial and temporal fungal colonization in roots, and both advantages and caveats of each technique should be considered in the subsequent interpretation of ecological patterns. Finally, including the isotopic signatures of root-associated fungi in the context of mycorrhizal symbiosis contributes to a direct observation of fungal participation to organic matter gain of the plant. The authors thank Christine Tiroch, Carina Bauer and Petra Eckert (BayCEER – Laboratory of Isotope Biogeochemistry) for skilful technical assistance with stable isotope abundance measurements, and also Johanna Pausch for allowing to use her laboratory for fungal extractions. Financial support was provided by an EMBO short-term fellowship to SIFG. The authors acknowledge the authorization by the Regierung von Oberfranken to collect samples of protected orchid species. Open Access funding enabled and organized by Projekt DEAL. None declared. SIFG and GG designed the research and collected the orchid material. PG and SK guided the fungal hyphal extraction by SIFG. CH and KS collected the arbuscular mycorrhizal plant material. GG supervised the isotope abundance analyses. SIFG performed the molecular analysis, analysed the data, and together with GG wrote the manuscript. All authors commented and approved the final version of the manuscript. Isotope abundance data are available in the Supporting Information. Raw sequencing data are available in GenBank/SRA under project number PRJNA966927. Methods S1 Methods used in this paper. Table S1 Isotope raw data and identity of reference plants at genus level. Please note: Wiley is 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. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

This study aimed to investigate the influence of genetic variants in neuroplasticity-related genes on antidepressant treatment phenotypes. The BDNF-TrkB signaling pathway, as well as the downstream kinases Akt and ERK and the mTOR pathway, have been implicated in depression and neuroplasticity. However, clinicians still struggle with the unpredictability of antidepressant responses in depressed patients. We genotyped 26 polymorphisms in BDNF, NTRK2, NGFR, CREB1, GSK3B, AKT, MAPK1, MTOR, PTEN, ARC, and SYN1 in 80 patients with major depressive disorder treated according to the Texas Medical Algorithm for 27 months at Hospital Magalhães Lemos, Porto, Portugal. Our results showed that BDNF rs6265, PTEN rs12569998, and SYN1 rs1142636 SNP were associated with treatment-resistant depression (TRD). Additionally, MAPK1 rs6928 and GSK3B rs6438552 gene polymorphisms were associated with relapse. Moreover, we found a link between the rs6928 MAPK1 polymorphism and time to relapse. These findings suggest that the BDNF, PTEN, and SYN1 genes may play a role in the development of TRD, while MAPK1 and GSK3B may be associated with relapse. GO analysis revealed enrichment in synaptic and trans-synaptic transmission pathways and glutamate receptor activity with TRD-associated genes. Genetic variants in these genes could potentially be incorporated into predictive models of antidepressant response.

Quantifying the abundances of fungi is key to understanding natural variation in mycorrhizal communities in relation to plant ecophysiology and environmental heterogeneity. High-throughput metabarcoding approaches have transformed our ability to characterize and compare complex mycorrhizal communities. However, it remains unclear how well metabarcoding read counts correlate with actual read abundances in the sample, potentially limiting their use as a proxy for species abundances.Here, we use droplet digital PCR (ddPCR) to evaluate the reliability of ITS2 metabarcoding data for quantitative assessments of mycorrhizal communities in the orchid species Neottia ovata sampled at multiple sites. We performed specific ddPCR assays for eight families of orchid mycorrhizal fungi and compared the results with read counts obtained from metabarcoding.Our results demonstrate a significant correlation between DNA copy numbers measured by ddPCR assays and metabarcoding read counts of major mycorrhizal partners of N. ovata, highlighting the usefulness of metabarcoding for quantifying the abundance of orchid mycorrhizal fungi. Yet, the levels of correlation between the two methods and the numbers of false zero values varied across fungal families, which warrants cautious evaluation of the reliability of low-abundance families.This study underscores the potential of metabarcoding data for more quantitative analyses of mycorrhizal communities and presents practical workflows for metabarcoding and ddPCR to achieve a more comprehensive understanding of orchid mycorrhizal communities.