Ectomycorrhizal fungi (EcMF) are key components of temperate ecosystems, and recent studies suggest that they can also inhabit non-EcM plant roots as endophytes. We aimed to (1) provide new evidence of EcMF colonization of non-EcM hosts, (2) offer direct microscopic confirmation of such endophytism and (3) assess factors influencing colonization, including plant identity, site and potential root filtering. Using ITS2 amplicon sequencing, we characterized root fungal communities in 66 non-EcM plant species sampled across nine French sites. At two sites, we compared rhizosphere and root communities to test for a root filtering. We also investigated endophytism in Russula species at two Russula-rich sites via FISH and confocal microscopy. We found substantial but variable proportions of EcMF sequences in non-EcM roots, particularly near EcM trees, indicating that endophytism may serve as a secondary ecological niche. Although EcMF were more abundant in the rhizosphere than in roots, community composition was similar, suggesting limited root filtering. Microscopy revealed metabolically active Russula hyphae within the apoplast of two non-EcM plant species. Our results confirm a dual EcM/endophyte niche for Russula spp., echoing patterns observed in other EcMF. The ecological implications of this duality remain to be explored.

ABSTRACT Truffles in the genus Tuber (Pezizales) are among the few ectomycorrhizal taxa successfully cultivated worldwide. Australia has recently become one of the top producers of Tuber melanosporum, a high-valued black truffle native to Europe. Truffle co-introductions in the Southern Hemisphere with their Northern Hemisphere plant symbionts are hypothesized to benefit from reduced ectomycorrhizal competition and natural enemies. In this study, we tested whether T. melanosporum in Australia experiences competition release by sampling soils and truffles across France, Spain, Italy, and Australia. Fungal and prokaryotic communities in truffle plantations were assessed in native (European) and non-native (Australian) habitats through ITS and 16S rDNA amplicon sequencing from soil and truffles. Community composition was primarily structured by site of origin and secondarily by presence of brûlé, vegetation-free area induced by truffle production of plant-growth inhibiting compounds, with significant interactions indicating site-dependent brûlé effects. European soils showed higher fungal richness outside the brûlé, with higher evenness overall and for ectomycorrhizal fungi only outside brûlé. T. melanosporum showed higher abundance in Australia, with significant differences restricted to outside the brûlé. Overall, ectomycorrhizal fungi in European soils had more than four times the taxa and higher diversity compared to Australian soils. Among the main competitors, Tomentella, Inocybe, and Trichophaea co-dominated in Europe, versus Scleroderma, Hebeloma, and Tarzetta in Australia. Despite differences in soil microbiomes, bacterial communities within T. melanosporum truffle ascocarps were strikingly similar across sites and continents and were dominated by Bradyrhizobium. Despite high site-level variation, our results support the competition release hypothesis, with reduced enemies benefiting T. melanosporum colonization outside the brûlé in Australia.IMPORTANCEThis study provides the first cross-hemisphere analysis of the truffle microbiome, comparing native and non-native soils and truffles from Europe and Australia. We demonstrate that the remarkable success of Tuber melanosporum cultivation in Australia is compatible with ecological release from competitors, which favors its development outside the brûlé. At the same time, we reveal striking cross-hemispheric similarities in truffle-associated bacterial communities, consistently dominated by Bradyrhizobium. These findings highlight both the novelty and transcontinental relevance of our work, offering new perspectives on fungal ecology and truffle cultivation.

Ectomycorrhizal fungi (EMF) colonize roots to establish symbiotic associations with plants. Sporocarps of the EMF Tuber spp. are considered as a delicacy in numerous countries and is a kind of EMF of great economic and social importance. Elucidating host responses to Tuber colonization would facilitate the exploration of symbiotic interactions and contribute to truffle cultivation. Tuber indicum and T. panzhihuanense, two primary commercial truffle species in China, were selected to colonize Pinus armandii and Carya illinoinensis in a two-and-a-half-year symbiosis experiment. Host performances, including growth, nutrient uptake, and physiological characteristics, were dynamically monitored. The molecular response of host leaf to Tuber symbiosis was further analyzed using RNA-seq. Tuber indicum and T. panzhihuanense exhibited superior colonization of P. armandii compared to that of C. illinoinensis. Both Tuber species enhanced the performance of the two hosts by increasing their height, stem circumference, and biomass. Phosphorus levels and activities of peroxidase and catalase in hosts were observed to increase during Tuber symbiosis. The results confirmed that Tuber colonization led to significant alterations in leaf transcriptomic profiles of the two trees. Tuber indicum and T. panzhihuanense both elicited defense-related regulation in host leaves, such as secondary metabolism, cell wall biogenesis, plant hormone signal transduction, and plant-pathogen interaction, with distinct patterns in P. armandii and C. illinoinensis. Our study provides an evaluation of host performance during truffle symbiosis and highlights the diverse patterns of Tuber-induced systematic defense regulation in hosts, offering insights into the specific symbiotic traits of Tuber-host pairs.

Tuber magnatum Picco (the Italian white truffle) is the most valuable and widely appreciated truffle. It is an ectomycorrhizal fungus known to associate with many broadleaf tree species. However, its mycorrhizae are rarely observed in the field, suggesting possible alternative symbiotic strategies, such as endophytism with non-ectomycorrhizal plants. In order to test potential endophytic interactions of T. magnatum with wild plants, a combination of polymerase chain reaction (PCR) and Fluorescence In Situ Hybridization (FISH) approaches were used. Specific FISH probes for T. magnatum were designed, tested in vitro on hyphae and/or ectomycorrhizae, and selected for their specificity. These probes were then used on a wide variety root samples of wild plants collected from three T. magnatum production areas in Italy and previously tested for the presence of T. magnatum mycelium using PCR-specific primers. Molecular analyses detected the presence of T. magnatum in 21 of 100 plant samples analyzed. FISH analysis confirmed the extracellular presence of active T. magnatum hyphae inside the root system of Carex pendula Huds plant. This study provides the first evidence of T. magnatum acting as an endophyte in an herbaceous plant. The newly designed, highly specific T. magnatum FISH probes can be used for further investigations to confirm the endophytic tendencies of T. magnatum and to understand their influence on the life cycle and biology of this fungus.

Background: The assembly of tree microbiota is a dynamic process that varies across space and time, influenced by both biotic and abiotic factors. Plants have developed defence strategies, mediated by phytohormones such as ethylene, to manage its interactions with pathogens and, in certain non-perennials, interactions with the commensal microbiota. However, whether and how ethylene regulation affects the assembly of tree microbiota is unknown. Results: We investigated the assembly of fungal and bacterial communities in poplars (P. tremula x. P. tremuloides T89), altered either in ethylene biosynthesis (ACO1), or in ethylene perception (etr1.1), by combining high-throughput amplicon sequencing with confocal microscopy. In parallel, we characterised root exudates, as well as root and shoot metabolomes of the different poplar lines grown on sterile soil and in the presence of microbiota using GC-MS. Alteration of ethylene levels had little impact on the metabolome of sterile poplars, but led to differential primary and secondary metabolic responses in the root and shoots of poplars colonised by microorganisms. These metabolomic changes were associated with a decrease in fungal colonisation of shoots, particularly by saprophytes in the early stages, whereas reduced ethylene stimulated root colonisation by fungi. Conversely, arbuscular mycorrhizal fungal and bacterial communities, as well as root exudation, were little affected by changes in ethylene production. Conclusion: The findings of this study suggest a potential dual role for ethylene in poplar, whereby its levels may either promote or inhibit microbial growth and activity, depending on the concentration, microbial trophic guild, and poplar organ. ### Competing Interest Statement The authors have declared no competing interest.

### Competing Interest Statement The authors have declared no competing interest.

Trees are associated with a broad range of microorganisms colonising the diverse tissues of their host. However, the early dynamics of the microbiota assembly microbiota from the root to shoot axis and how it is linked to root exudates and metabolite contents of tissues remain unclear. Here, we characterised how fungal and bacterial communities are altering root exudates as well as root and shoot metabolomes in parallel with their establishment in poplar cuttings (Populus tremula x tremuloides clone T89) over 30 days of growth. Sterile poplar cuttings were planted in natural or gamma irradiated soils. Bulk and rhizospheric soils, root and shoot tissues were collected from day 1 to day 30 to track the dynamic changes of fungal and bacterial communities in the different habitats by DNA metabarcoding. Root exudates and root and shoot metabolites were analysed in parallel by gas chromatography-mass spectrometry. Our study reveals that microbial colonisation triggered rapid and substantial alterations in both the composition and quantity of root exudates, with over 70 metabolites exclusively identified in remarkably high abundances in the absence of microorganisms. Noteworthy among these were lipid-related metabolites and defence compounds. The microbial colonisation of both roots and shoots exhibited a similar dynamic response, initially involving saprophytic microorganisms and later transitioning to endophytes and symbionts. Key constituents of the shoot microbiota were also discernible at earlier time points in the rhizosphere and roots, indicating that the soil constituted a primary source for shoot microbiota. Furthermore, the microbial colonisation of belowground and aerial compartments induced a reconfiguration of plant metabolism. Specifically, microbial colonisation predominantly instigated alterations in primary metabolism in roots, while in shoots, it primarily influenced defence metabolism. This study highlighted the profound impact of microbial interactions on metabolic pathways of plants, shedding light on the intricate interplay between plants and their associated microbial communities.

The study of the dialogue between microorganisms at the molecular level is becoming essential to understand their relationship (antagonist, neutral, or beneficial interactions) and its impact on the organization of the microbial community. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption/ionization (MALDI) is a technique that reveals the spatial distribution of molecules on a sample surface that may be involved in interactions between organisms. An experimental limitation to perform MALDI MSI is a flat sample surface, which in many cases could not be achieved for bacterial colonies such as filamentous bacteria (e.g., Streptomyces). In addition, sample heterogeneity affects sample dryness and MALDI matrix deposition prior to MSI. To avoid such problems, we introduce an additional step in the sample preparation. A polymeric membrane is interposed between the microorganisms and the agar-based culture medium, allowing the removal of bacterial colonies prior to MSI of the homogeneous culture medium. A proof of concept was evaluated on Streptomyces ambofaciens (a soil bacterium) cultures on solid media. As the mycelium was removed at the same time as the polymeric membrane, the metabolites released into the medium were spatially resolved by MALDI MSI. In addition, extraction of the recovered mycelium from the membrane confirmed the identification of the metabolites by ESI MS/MS analysis. This approach allows both the spatial distribution of metabolites produced by microorganisms in an agar medium to be studied under well-controlled sample preparation and their structure to be elucidated. This capability is illustrated using desferrioxamine E, a siderophore produced by S. ambofaciens.

Despite lignin being a key component of wood, the dynamics of tracheid lignification are generally overlooked in xylogenesis studies, which hampers our understanding of environmental drivers and blurs the interpretation of isotopic and anatomical signals stored in tree rings. Here, we analyzed cell wall formation in silver fir (Abies alba Mill.) tracheids to determine if cell wall lignification lags behind secondary wall deposition. For this purpose, we applied a multimodal imaging approach combining transmitted light microscopy (TLM), confocal laser scanning microscopy (CLSM), and confocal Raman microspectroscopy (RMS) on anatomical sections of wood microcores collected in northeast France on 11 dates during the 2010 growing season. Wood autofluorescence after laser excitation at 405 and 488 nm associated with the RMS scattering of lignin and cellulose, respectively, which allowed identification of lignifying cells (cells showing lignified and nonlignified wall fractions at the same time) in CLSM images. The number of lignifying cells in CLSM images mirrored the number of wall-thickening birefringent cells in polarized TLM images, revealing highly synchronized kinetics for wall thickening and lignification (similar timings and durations at the cell level). CLSM images and RMS chemical maps revealed a substantial incorporation of lignin into the wall at early stages of secondary wall deposition. Our results show that most of the cellulose and lignin contained in the cell wall undergo concurrent periods of deposition. This suggests a strong synchronization between cellulose and lignin-related features in conifer tree-ring records, as they originated over highly overlapped time frames.

Copper-azole based formulations have been widely used to protect wood timbers against fungal decay. While these treatments are efficient for wood protection, leaching of both copper and azoles into the environment has deleterious impact on soils and surface waters. No bioremediation process is currently available for disposable of these wood wastes. Exploiting the natural ability of certain fungi to tolerate these active compounds, we propose that some ligninolytic fungi could serve as effective biocatalysts for detoxifying copper-azole formulations. Using the white-rot fungus Phanerochaete chrysosporium as a model, we demonstrated that these fungi engage multiple strategies to counteract the antifungal effect of azoles present in the preservatives. These include the modulation of the lipids and sterols content, the maintenance of DNA integrity, detoxification of azoles by extracellular degradation likely through the Fenton chemistry, biosorption at the cell wall, efflux, and intracellular detoxification by the three-step detoxification pathway. By using comparative transcriptomics between a copper-azole formulation and a copper-quaternary ammonium formulation, we identified genes specifically involved in azole resistance and detoxification within this complex system. This opens new perspectives for managing azoles residues through mycoremediation processes.

### Competing Interest StatementThe authors have declared no competing interest.

Riparian forests remain largely understudied in the context of climate change in comparison to other forest ecosystems although they serve multiple socio-ecological functions. We evaluated local adaptation and adaptive potential in Populus nigra L., an emblematic tree species of European riparian forests. We set up a reciprocal transplant experiment and measured 17 structural and functional traits among 10 progenies of two genetically differentiated populations. The populations originated from two separate watersheds differing in climate conditions and a total of 1200 seedlings were grown in 1-m3 mesocosms at both sites for one growing season. Traits measured were related to growth, leaf physiology and xylem water transport. The populations showed similar biomass suggesting no local adaptation for overall performance but displayed distinct trait syndromes and plastic abilities. The southern population primarily adjusted through changes in allocation and leaf water-use efficiency while the northern population primarily adjusted through changes in specific leaf area. Genetic variation within populations was most of the times equal or larger than between populations. This combined with the generally moderate to high heritability values and the observed plasticity suggested significant adaptive potential. Altogether, our findings reveal that although evolution may not lead to obvious differentiation between populations in global performance, integrated multi-trait approaches are highly valuable to shed light on how evolution may shape distinct underlying functional strategies among populations resulting in a similar outcome.

Fungi are the principal decomposers of wood together with xylophage insects and, as such, have a central role in nutrient cycling of forest ecosystems. These fungi are also envisaged as promising tools for converting wood and waste of wood industries into chemicals, as alternative to fossil chemicals. At the same time, wood decomposers pose a threat to wooden building materials and are intensively fought. As a consequence, intense researches have been conducted over the past 50 years to identify the fungi responsible for wood decomposition, the mechanisms by which they do so, the wood properties involved in resistance or sensitivity to attacks and ways to preserve woods. Many tools are now available to study fungal colonization of wood, including: "omics" techniques, enzymatic assays, spectrometry, etc. However, all these approaches provide bulk information and the data obtained by these methods contain no information on the localization of fungi, the stage of decomposition of the wood and the potential interactions between microorganisms. In these regards, microscopy approaches provide complementary information that can strengthen conclusions. The present chapter describes a diverse range of microscopy approaches, from simple bench light microscopy to confocal and electron microscopies, to shed light on the way fungi colonize wood tissues.

Bacteria can live in a variety of interkingdom communities playing key ecological roles. The microbiome of leaf-cutting attine ant colonies are a remarkable example of such communities, as they support ants’ metabolic processes and the maintenance of ant-fungus gardens. Studies on this topic have explored the bacterial community of the whole fungus garden, without discerning bacterial groups associated with the nutrient storage structures (gongylidia) of ant fungal cultivars. Here we studied bacteria isolated from the surface of gongylidia in the cultivars of Atta sexdens and Acromyrmex coronatus , to assess whether the bacterial community influences the biology of the fungus. A total of 10 bacterial strains were isolated from gongylidia ( Bacillus sp., Lysinibacillus sp., Niallia sp., Staphylococcus sp., Paenibacillus sp., Pantoea sp., Staphylococcus sp., and one Actinobacteria). Some bacterial isolates increased gongylidia production and fungal biomass while others had inhibitory effects. Eight bacterial strains were confirmed to form biofilm-like structures on the fungal cultivar hyphae. They also showed auxiliary metabolic functions useful for the development of the fungal garden such as phosphate solubilization, siderophore production, cellulose and chitin degradation, and antifungal activity against antagonists of the fungal cultivar. Bacteria-bacteria interaction assays revealed heterogeneous behaviors including synergism and competition, which might contribute to regulate the community structure inside the garden. Our results suggest that bacteria and the ant fungal cultivar interact directly, across a continuum of positive and negative interactions within the community. These complex relationships could ultimately contribute to the stability of the ant-fungus mutualism.

Plants interact with a broad range of microorganisms, such as bacteria and fungi. In plant roots, complex microbial communities participate in plant nutrition and development as well as in the protection against stresses. The establishment of the root microbiota is a dynamic process in space and time regulated by abiotic (e.g., edaphic, climate, etc.) and biotic factors (e.g., host genotype, root exudates, etc.). In the last 20 years, the development of metabarcoding surveys, based on high-throughput next-generation sequencing methods, identified the main drivers of microbial community structuration. However, identification of plant-associated microbes by sequencing should be complemented by imaging techniques to provide information on the micrometric spatial organization and its impact on plant-fungal and fungal-fungal interactions. Laser scanning confocal microscopy can provide both types of information and is now used to investigate communities of endophytic, endomycorrhizal, and ectomycorrhizal fungi. In this chapter, we present a protocol enabling the detection of fungal individuals and communities associated to the plant root system.

Copper-based formulations of wood preservatives are widely used in industry to protect wood materials from degradation caused by fungi. Wood treated with preservatives generate toxic waste that currently cannot be properly recycled. Despite copper being very efficient as an antifungal agent against most fungi, some species are able to cope with these high metal concentrations. This is the case for the brown-rot fungus Rhodonia placenta and the white-rot fungus Phanerochaete chrysosporium, which are able to grow efficiently in pine wood treated with Tanalith E3474. Here, we aimed to test the abilities of the two fungi to cope with copper in this toxic environment and to decontaminate Tanalith E-treated wood. A microcosm allowing the growth of the fungi on industrially treated pine wood was designed, and the distribution of copper between mycelium and wood was analysed within the embedded hyphae and wood particles using coupled X-ray fluorescence spectroscopy and Scanning Electron Microscopy (SEM)/Electron Dispersive Spectroscopy (EDS). The results demonstrate the copper biosorption capacities of P. chrysosporium and the production of copper-oxalate crystals by R. placenta. These data coupled to genomic analysis suggest the involvement of additional mechanisms for copper tolerance in these rot fungi that are likely related to copper transport (import, export, or vacuolar sequestration).

SummaryThe factors that vary the aroma ofTuber magnatumfruiting bodies are poorly understood. The study determined the headspace aroma composition, sensory aroma profiles, maturity, and microbiome composition fromT. magnatumoriginating from Italy, Croatia, Hungary, and Serbia, and tested if truffle aroma is dependent on provenance and if fruiting body volatiles are explained by maturity and/or microbiome composition.Headspace volatile profiles were determined by gas chromatography-mass spectrometry-olfactometry (GC-MS-O) and aroma of fruiting body extracts were sensorially assessed. Fruiting body maturity were estimated through spore melanisation. Bacterial community was determined using 16S rRNA amplicon sequencing.Main odour active compounds were present in all truffles but varied in concentration. Aroma of truffle extracts were sensorially discriminated by sites. However, volatile profiles of individual fruiting bodies varied more within sites than across geographic area, while maturity level did not play a role. Microbiome composition varied highly and was partially explained by provenance. A few rare bacterial operational taxonomical units associated with select few non-odour active volatile compounds.Specificities of the aroma ofT. magnatumtruffles are more likely linked to individual properties than provenance. Some constituents of the microbiome may provide biomarkers of provenance and be linked to non-odour active volatiles.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells. While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

The tree root microbiome is composed of a very diverse set of bacterial and fungal communities. These microorganisms have a profound impact on tree growth, development, and protection against different types of stress.