Maaria Rosenkranz

Although trees encounter diverse soil‐associated fungi, it is unclear how they adjust their physiology in response to volatile organic compounds (VOCs) from fungi with contrasting ecological strategies before physical contact. Here, we tested whether VOCs emitted by fungi are sufficient to induce systemic, fungus‐dependent metabolic responses in poplar. Populus × canescens roots were exposed to VOCs from a pathogen ( Heterobasidion annosum ), a saprotroph ( Postia placenta ), or an ectomycorrhizal mutualist ( Laccaria bicolor ) for 6 weeks in a contact‐free pot‐in‐pot system. Untargeted LC–MS metabolomics was used to characterize VOC‐induced metabolic reprogramming in roots and leaves. Fungal VOC exposure alone reconfigured root and leaf metabolomes, with both shared and fungus‐dependent responses despite VOC exposure being restricted to the belowground compartment. Treatment‐associated differences varied between tissues. H. annosum was associated with pronounced suppression of high‐confidence features in leaves but a more balanced response in roots. P. placenta showed several uniquely upregulated root features together with selected lipid‐associated changes, and L. bicolor produced comparatively few unique high‐confidence changes. These results show that poplar can respond systemically and differentially to VOCs from contrasting fungal species before physical contact, while the extent to which these responses can be generalised across fungal lifestyles remains to be established.

Plants are exposed to complex interactions with belowground organisms, yet how they differentiate between mutualistic and pathogenic fungi before physical contact remains largely unknown. We exposed the roots of young Populus × canescens (Aiton) Sm. plants to volatile organic compounds (VOCs) emitted by fungi with contrasting lifestyles: the pathogenic Heterobasidion annosum (Fr.) Bref., the saprotrophPostia placenta (Fr.) M.J. Larsen & Lombard and the ectomycorrhizal fungus Laccaria bicolor (Maire) P.D. Orton. Volatile organic compound analysis of the shared rhizosphere headspace and leaf emissions revealed that poplar plants could perceive and respond to fungal identity solely through airborne cues. The root-zone headspace contained fungus-specific sesquiterpene fingerprints that remained similar after 3 and 6 weeks of co-cultivation: Pathogen-derived VOCs induced constantly high sesquiterpene emissions from the root-zone, whereas mycorrhizal VOCs caused low but targeted emissions of specific sesquiterpenes. In contrast, saprotrophic VOCs caused a temporal shift in root-zone VOC pattern, with increased sesquiterpene emissions after 6 weeks. Fungal VOC exposure also altered leaf VOC emissions, enriching alkanes, esters and monoterpenes. Initially, leaf VOC emissions were fungal lifestyle-specific but they converged over time, indicating systemic signal integration of belowground signals. These findings demonstrate that trees can discriminate ‘friend-versus-foe’ through VOCs alone, extending pattern-recognition theory beyond contact-dependent cues. Multivariate analyses suggested organ-specific chemical strategies: roots function as chemosensors decoding fungal volatilomes, while systemic adjustments shape aboveground VOC profiles. Understanding the plant response to fungal VOCs may offer potential for developing early pathogen diagnostics and further elucidate the volatile-mediated plant–fungal interactions.

Summary: Rationale: Although trees encounter diverse fungal communities, it is unclear how they adjust their physiology in response to fungal ecological strategies before physical contact. We tested whether volatile organic compounds (VOCs) emitted by fungi are sufficient to induce systemic, lifestyle-consistent metabolic states in poplar roots and leaves. Methods: Populus × canescens roots were exposed to VOCs from a pathogen ( Heterobasidion annosum ), a saprotroph ( Postia placenta ) or an ectomycorrhizal mutualist ( Laccaria bicolor ) for six weeks in a contact-free pot-in-pot system. Untargeted LC–MS metabolomics characterized VOC-induced metabolic reprogramming in roots and leaves. Key results: Fungal VOC exposure alone reconfigured the metabolomes of roots and leaves, with strong discrimination between treatments despite belowground exposure. Poplar revealed a shared VOC-responsive component, but also fungus-specific programmes: pathogen VOCs produced a suppression-dominated systemic phenotype; saprotroph VOCs promoted lipid-centred metabolic activation; and mutualist VOCs elicited restrained, compatibility-consistent shifts with targeted pathway modulation. Main conclusion: Volatile-mediated surveillance allows trees to anticipate fungal lifestyle-associated cues and adjust systemic metabolism before physical contact occurs. This links airborne fungal cues to whole-plant physiological configuration and extends plant–fungal recognition beyond contact-dependent mechanisms. One-sentence summary: Volatile-mediated surveillance allows trees to anticipate fungal lifestyle and adjust systemic metabolism before physical contact occurs.

Plants are exposed to complex interactions with belowground organisms, yet how they differentiate between mutualistic and pathogenic fungi before physical contact remains largely unknown. We exposed the roots of young Populus × canescens to volatile organic compounds (VOCs) emitted by either a pathogenic ( Heterobasidion annosum ), a saprotrophic ( Postia placenta ), or an ectomycorrhizal ( Laccaria bicolor ) fungus. VOC analysis of the shared rhizosphere headspace and leaf emissions revealed that poplar plants could perceive and respond to fungal identity solely through airborne cues. The root-zone headspace contained fungus-specific sesquiterpene fingerprints that remained similar after three and six weeks of co-cultivation: Pathogen-derived VOCs induced constant high sesquiterpene emissions from the root-zone, whereas mycorrhiza caused low but targeted emissions of specific sesquiterpenes. In contrast, saprotrophic VOCs caused a temporal shift in root-zone VOC pattern, with increased sesquiterpene emissions after six weeks. Fungal VOC exposure also altered leaf VOC emissions, enriching alkanes, esters and monoterpenes. Initially, leaf VOC emissions were fungal lifestyle-specific but they converged over time, indicating systemic signal integration of belowground signals. These findings demonstrate that trees can discriminate “friend-versus-foe” through VOCs alone, extending pattern-recognition theory beyond contact-dependent cues. Multivariate analyses suggested organ-specific chemical strategies: roots function as chemosensors decoding fungal volatilomes, while systemic adjustments shape aboveground VOC profiles. Understanding the plant response to fungal VOCs may offer potential for developing early pathogen diagnostics and further elucidate the volatile-mediated plant-fungal interactions.

Isoprene, the most abundant biogenic hydrocarbon in the atmosphere, is known to protect photosynthesis from abiotic stress and significantly impact atmospheric chemistry. While laboratory studies show that isoprene can enhance plant immunity, its role in plant‐plant communication under natural field conditions remains unclear. In a 2‐year field experiment, we used wild‐type and transgenic silver birch ( Betula pendula ) lines with enhanced isoprene emission levels to examine their impact on neighboring Arabidopsis thaliana , including wild‐type and immune signaling mutants ( llp1: legume lectin‐like protein 1; jar1: jasmonate resistant 1 ). Receiver plants exposed to higher isoprene levels showed increased resistance to Pseudomonas syringae , independent of jasmonate signaling but dependent on LLP1, a protein essential for systemic acquired resistance. Volatile analysis indicated isoprene as an airborne molecule that can also trigger an immune response in neighboring plants along with other terpenoids. Our study using transgenic birches in a complex environment provides new insights into the molecular mechanisms underlying plant volatile perception and expands our understanding of plant chemical communication in terrestrial ecosystems.

The genus Trichoderma (Hypocreaceae, Ascomycota) compromises over 400 known species, that are found in various soils, on plant surfaces and as plant endophytes. Interactions between the mycoparasitic Trichoderma spp. and beneficial ectomycorrhizal fungi such as Laccaria bicolor (Hydnangiaceae, Basidiomycota) can influence the structure of fungal communities and plant symbioses. In this study, we conducted in vitro dual-culture experiments involving L. bicolor and four Trichoderma strains (T. harzianum WM24a1, MS8a1, ES8g1, and T. atrobrunneum) to analyze their metabolic responses in relation to varying degrees of physical contact. Using integrated analyses of volatile organic compounds (VOCs), hyphal metabolomes, and secreted exudates, we uncovered strong contact- and strain-dependent growth inhibition patterns: Trichoderma growth was suppressed under shared headspace, whereas L. bicolor was more strongly inhibited under direct contact. Metabolomic profiling revealed distinct and strain-specific alterations in both VOC and soluble metabolite profiles during co-cultivation, with hundreds of discriminant mass features affected. Key metabolic pathways, including amino acid, carbohydrate, lipid, and secondary metabolite biosynthesis, showed differential enrichment depending on the interaction stage and fungal partner. These results demonstrate that Trichoderma–Laccaria interactions are mediated by dynamic, contact-specific chemical reprogramming and suggest that fungal recognition and competition involve coordinated changes in both volatile and non-volatile metabolite production. Our findings provide a foundation for exploring how such antagonistic interactions may influence tripartite communication in plant-associated microbial networks. They also highlight the potential role of both emitted and secreted fungal metabolites in shaping interaction dynamics through putative non-self-recognition mechanisms.

Isoprene, the most abundant hydrocarbon emitted by vegetation, protects photosynthesis against oxidative and thermal stress and significantly impacts atmospheric chemistry. While laboratory studies suggest a role for isoprene in plant defense, its function in plant-plant communication under natural conditions remains unclear. Here, we demonstrate that isoprene acts as a signaling molecule, triggering systemic immune responses in neighboring plants against bacterial pathogens under field conditions. We established an experimental system using wild-type and transgenic silver birch ( Betula pendula ) lines engineered to emit varying levels of isoprene. Over two growing seasons, we examined the effects of birch-emitted volatile organic compounds on neighboring Arabidopsis thaliana, including wild-type and immune signaling mutants ( llp1 : legume lectin-like protein 1; jar1 : jasmonate resistant 1). Isoprene emission rates positively correlated with systemic resistance in receiver plants, with higher emissions enhancing inhibition of Pseudomonas syringae growth. This immune response occurred independently of jasmonate signaling but required functional LLP1, suggesting a specific recognition pathway. By combining Arabidopsis mutant responses with birch volatile profiles, we confirmed that isoprene, rather than other terpenoids, mediates this effect. These findings reveal an unrecognized ecological function of isoprene as an airborne immune signal, establishing transgenic birch as a robust model for studying volatile-mediated plant defense networks in natural environments. Our results provide new insights into the molecular mechanisms underlying plant volatile perception and expand our understanding of chemical communication in terrestrial ecosystems. Significance Statement: Isoprene, the most abundant volatile organic compound released by plants, protects against abiotic stress. Here, we demonstrate that isoprene also functions under ambient atmosphere as a short-distance immune signal, enhancing neighboring plants’ resistance to bacterial pathogens. This finding reveals a putative ecological role of isoprene in plant defense networks and suggests strategies for sustainable agriculture and forest management under environmental stress.

With over 250 known species, the genus Trichoderma (Ascomycota, Hypocreaceae) is found in various soils, on plant surfaces and as plant endophytes. While Trichoderma species are known as mycoparasites, their antagonistic behaviour can also negatively affect other beneficial fungi, such as mycorrhizal fungi. To gain insight into the metabolic signals involved in the interactions between the ectomycorrhizal fungus (ECM) Laccaria bicolor (Basidomycota, Hydnangiaceae), and different mycoparasitic Trichoderma spp. (T. harzianum strains WM24a1, MS8a1 and ES8g1, and T. atrobrunneum), we performed in vitro dual-confrontation experiments. We studied the volatile organic compounds (VOCs), hyphal metabolomes and soluble metabolites released by each of the fungi in various co-cultivation scenarios. The results revealed an altered growth of the mycelia depending on the degree of contact: When Trichoderma spp. and L. bicolor shared only the same headspace, Trichoderma spp. growth was at least partially inhibited, whereas in direct contact the growth of L. bicolor was impaired. Distinct strain- and species-specific changes in hyphal metabolites, in exudates and volatile emission were revealed from each of the studied fungi. We identified both core metabolite profiles and interaction-specific metabolic responses that were related to carbohydrate, lipid, nucleotide, energy and amino acid metabolisms. Volatile and soluble metabolites revealed temporal and spatial adjustments in dual cultures compared to solitary cultures, suggesting rapid contact-dependent adaptations and demonstrating the dynamic communication mechanisms between Trichoderma spp. and the ECM. These results suggest a central role for both emitted and secreted fungal metabolites in the fungal non-self-recognition and in interaction with each other.

Summary: Trichoderma spp. are gaining popularity in agriculture and forestry due to their multifaceted roles in promoting plant growth through e.g. nutrient translocation, hormone production, induction of plant systemic resistance, but also direct antagonism of other fungi. However, the mycotrophic nature of the genus bears the risk of possible interference with other native plant-beneficial fungi, such as ectomycorrhiza, in the rhizosphere. Such interference could yield unpredictable consequences for the host plants of these ecosystems. We investigated whether Trichoderma spp. can differentiate between beneficial ectomycorrhizal fungi (represented by Laccaria bicolor and Hebeloma cylindrosporum ) and pathogenic fungi (represented by Fusarium graminearum and Alternaria alternata ) in different confrontation scenarios, including a newly developed olfactometer “race tube”-like system. Using two independent species, T. harzianum, and T. atrobrunneum , with plant-growth-promoting and immune-stimulating properties towards Populus x canescens , our study revealed robustly accelerated growth towards phytopathogens, while showing a contrary response to ectomycorrhizal fungi. Transcriptomic analyses identified distinct genetic programs during interaction corresponding to the lifestyles, emphasizing the expression of mycoparasitism-related genes only in the presence of phytopathogens. The findings reveal a critical mode of fungal community interactions belowground and suggest that Trichoderma spp. can distinguish between fungal partners of different lifestyles already at a distance.

Metabolomics studies are becoming increasingly common for understanding how plant metabolism responds to changes in environmental conditions, genetic manipulations, and treatments. Despite the recent advances in metabolomics workflow, the sample preparation process still limits the high-throughput analysis in large-scale studies. Here, we present a highly flexible robotic system that integrates liquid handling, sonication, centrifugation, solvent evaporation, and sample transfer processed in 96-well plates to automatize the metabolite extraction from leaf samples. We transferred an established manual extraction protocol performed to a robotic system, and with this, we show the optimization steps required to improve reproducibility and obtain comparable results in terms of extraction efficiency and accuracy. We then tested the robotic system to analyze the metabolomes of wild-type and four transgenic silver birch (Betula pendula) lines under unstressed conditions. Birch trees were engineered to overexpress the poplar (Populus x canescens) isoprene synthase (PcISPS) and emit various amounts of isoprene. By fitting the different isoprene emission capacities of the transgenic trees with their leaf metabolomes, we observed an isoprene-dependent upregulation of some flavonoids and other secondary metabolites as well as carbohydrates, amino acid and lipid metabolites. In contrast, the disaccharide sucrose was found to be strongly negatively correlated to isoprene emission. The presented study illustrates the power of integrating robotics to increase sample throughput, reduce human errors and labor time, and ensure a fully controlled, monitored, and standardized sample preparation procedure. Due to its modular and flexible structure, the robotic system can be easily adapted to other extraction protocols for the analysis of various tissues or plant species to achieve high-throughput metabolomics in plant research.