The ectomycorrhizal community of urban linden trees in Gdansk, Poland.

The linden tree (Tilia spp.) is a popular tree for landscaping and urban environments in central and northwest European countries, and it is one of the most popular in cities in Poland. Ectomycorrhizal fungi form a symbiosis with many urban tree species and protect the host plant from heavy metals and against salinity. The aim of this study was to characterise the ECM fungal community of urban linden trees along the tree damage gradient. The study was performed on two sites located in the centre of the city of Gdansk, in northern Poland. The vitality assessment of urban linden trees was made according to Roloff's classification. Tree damage classes were related to soil characteristics using principal component analysis. The five ectomycorrhizal fungal species were shared among all four tree damage classes, and Cenococcum geophilum was found to be the most abundant and frequent ectomycorrhizal fungal species in each class. Soil samples collected in the vicinity of trees belonging to the R0 class had significantly lower pH Na, Cl and Pb content than other soils. Our knowledge of ectomycorrhizal communities in urban areas is still limited, and these findings provide new insights into ectomycorrhizal distribution patterns in urban areas.

Canopy Position Has a Stronger Effect than Tree Species Identity on Phyllosphere Bacterial Diversity in a Floodplain Hardwood Forest.

The phyllosphere is a challenging microbial habitat in which microorganisms can flourish on organic carbon released by plant leaves but are also exposed to harsh environmental conditions. Here, we assessed the relative importance of canopy position-top, mid, and bottom at a height between 31 and 20 m-and tree species identity for shaping the phyllosphere microbiome in a floodplain hardwood forest. Leaf material was sampled from three tree species-maple (Acer pseudoplatanus L.), oak (Quercus robur L.), and linden (Tilia cordata MILL.)-at the Leipzig canopy crane facility (Germany). Estimated bacterial species richness (Chao1) and bacterial abundances approximated by quantitative PCR of 16S rRNA genes exhibited clear vertical trends with a strong increase from the top to the mid and bottom position of the canopy. Thirty operational taxonomic units (OTUs) formed the core microbiome, which accounted for 77% of all sequence reads. These core OTUs showed contrasting trends in their vertical distribution within the canopy, pointing to different ecological preferences and tolerance to presumably more extreme conditions at the top position of the canopy. Co-occurrence analysis revealed distinct tree species-specific OTU networks, and 55-57% of the OTUs were unique to each tree species. Overall, the phyllosphere microbiome harbored surprisingly high fractions of Actinobacteria of up to 66%. Our results clearly demonstrate strong effects of the position in the canopy on phyllosphere bacterial communities in a floodplain hardwood forest and-in contrast to other temperate or tropical forests-a strong predominance of Actinobacteria.

Soil organic matter rather than ectomycorrhizal diversity is related to urban tree health.

Urban trees provide many ecosystem services, including carbon sequestration, air quality improvement, storm water attenuation and energy conservation, to people living in cities. Provisioning of ecosystem services by urban trees, however, may be jeopardized by the typically poor quality of the soils in urban areas. Given their well-known multifunctional role in forest ecosystems, ectomycorrhizal fungi (EcM) may also contribute to urban tree health and thus ecosystem service provisioning. Yet, no studies so far have directly related in situ EcM community composition to urban tree health indicators. Here, two previously collected datasets were combined: i) tree health data of 175 Tilia tomentosa trees from three European cities (Leuven, Strasbourg and Porto) estimated using a range of reflectance, chlorophyll fluorescence and physical leaf indicators, and ii) ectomycorrhizal diversity of these trees as characterized by next-generation sequencing. Tree health indicators were related to soil characteristics and EcM diversity using canonical redundancy analysis. Soil organic matter significantly explained variation in tree health indicators whereas no significant relation between mycorrhizal diversity variables and the tree health indicators was found. We conclude that mainly soil organic matter, through promoting soil aggregate formation and porosity, and thus indirectly tree water availability, positively affects the health of trees in urban areas. Our results suggest that urban planners should not overlook the importance of soil quality and its water holding capacity for the health of urban trees and potentially also for the ecosystem services they deliver. Further research should also study other soil microbiota which may independently, or in interaction with ectomycorrhiza, mediate tree performance in urban settings.

The genetic relationships within Apiognomonia errabunda and related species.

Species of Apiognomonia are some of the most ubiquitous leaf-associated fungi of broad-leaved trees of the northern temperate zone. Especially widespread and diverse is Apiognomonia errabunda, found mostly on beech, oak, and linden. This species and its closest relatives are known for their complicated phylogenetic relationships that have caused considerable confusion in the past. In the present paper, we describe the results of a three-gene-based reconstruction of phylogenetic relationships between A. errabunda, A. veneta, and A. hystrix for 373 isolates. Using combined information from the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) and intron regions of actin (ACT) and calmodulin (CAL) genes for 263 isolates, we confirmed the occurrence of host-associated lineages within A. errabunda. However, the clustering of isolates by host species was incomplete: some isolates occurred on the "wrong" hosts and a number of isolates carried mixed genetic profile, indicating substantial level of inter-host group recombination. In addition, a number of isolates were identified as putative, pending further verification, interspecies hybrids between the much more divergent A. errabunda and A. hystrix.

Effects of root and leaf litter identity and diversity on oribatid mite abundance, species richness and community composition.

Habitat heterogeneity is an important driver of aboveground species diversity but few studies have investigated effects on soil communities. Trees shape their surrounding by both leaf litter and roots generating small scale heterogeneity and potentially governing community patterns of soil organisms. To assess the role of vegetation for the soil fauna, we studied whether tree species (Fagus sylvatica L., Acer pseudoplatanus L., Fraxinus excelsior L., Tilia cordata Mill.), markedly differing in leaf litter quality and root associated mycorrhizal symbionts, affect oribatid mite communities by shaping below- and aboveground resources and habitat complexity and availability. Oribatid mite abundance, species richness, community structure and the proportion of litter living and parthenogenetic individuals were analyzed and related to microbial biomass and the amount of remaining litter mass. Although leaf litter species with higher nutritional values decomposed considerably faster, microbial biomass only slightly differed between leaf litter species. Neither root species nor leaf litter species affected abundance, species richness or community structure of oribatid mites. However, root species had an effect on the proportion of parthenogenetic individuals with increased proportions in the presence of beech roots. Overall, the results suggest that identity and diversity of vegetation via leaf litter or roots are of minor importance for structuring oribatid mite communities of a temperate forest ecosystem.

Leaf litter species identity influences biochemical composition of ectomycorrhizal fungi.

In forest ecosystems, ectomycorrhizal (ECM) fungi are important for plant growth and soil biogeochemical processes. The biochemical composition of ECM mycelium is an important fungal effect trait with consequences for its decomposition rate, and consequently on soil carbon pools and plant nutrition. Although the link between ECM fungi and leaf litter-released nutrients is well known, the response of ECM fungal biochemical composition to different leaf litter species remains poorly understood. To determine how leaf litter quality influences ECM fungi's biochemical profiles, we planted young beech trees in an oak forest and replaced the natural leaf litter with that of European beech (Fagus sylvatica), ash (Fraxinus excelsior), maple (Acer pseudoplatanus), or lime (Tilia cordata). We assessed the biochemical profiles of ECM root tips colonized by common fungal taxa in temperate forests (i.e., Cenococcum geophilum, Inocybe sp., and Lactarius subdulcis), using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). ECM fungal biochemical composition changed with leaf litter species. Changes were apparent in the infrared absorption bands assigned to functional groups of lipids, amides, and carbohydrates. C. geophilum and L. subdulcis exhibited large spectral differences corresponding to the initial pattern of leaf litter chemical composition between samples collected in the beech and ash leaf litter treatments. In contrast, Inocybe sp. was influenced by lime, but with no differences between samples from ash or beech leaf litter treatments. Although the spectral bands affected by leaf litter type differed among ECM fungi, they were mainly related to amides, indicating a dynamic response of the fungal proteome to soil nutritional changes. Overall, the results indicate that the biochemical response of ECM fungi to leaf litter species varies among ECM fungal species and suggests that the biochemical composition of ECM mycelium is a fungal response trait, sensitive to environmental changes such as shifts in leaf litter species.

Variation in ectomycorrhizal fungal communities associated with Silver linden (Tilia tomentosa) within and across urban areas.

Trees in urban areas face harsh environmental conditions. Ectomycorrhizal fungi (EcM) form a symbiosis with many tree species and provide a range of benefits to their host through their extraradical hyphal network. Although our understanding of the environmental drivers and large scale geographical variation of EcM communities in natural ecosystems is growing, our knowledge of EcM communities within and across urban areas is still limited. Here, we characterized EcM communities using Illumina miseq sequencing on 175 root samples of the urban tree Tilia tomentosa from three European cities, namely Leuven (Belgium), Strasbourg (France) and Porto (Portugal). We found strong differences in EcM richness and community composition between cities. Soil acidity, organic matter and moisture content were significantly associated with EcM community composition. In agreement, the explained variability in EcM communities was mostly attributed to general soil characteristics, whereas very little variation was explained by city and heavy metal pollution. Overall, our results suggest that EcM communities in urban areas are significantly associated with soil characteristics, while heavy metal pollution and biogeography had little or no impact. These findings deliver new insights into EcM distribution patterns in urban areas and contribute to specific inoculation strategies to improve urban tree vitality.

Antioxidative and antifungal response of woody species to environmental conditions in the urban area.

The complexity of ecological conditions in urban areas imposes the plant species need for the development of various biochemical and physiological adaptive strategies. The aim of our research was to examine the antioxidative and antifungal metabolism of species Pinus nigra, Picea omorika, Tilia cordata and Betula pendula from the area of Banja Luka City (urban area) during two vegetation seasons (spring and autumn) and compared with the same species from forest habitats. Changes in the protein concentration, activity and isoenzyme profiles of peroxidases (POD, EC 1.11.1.7), content and antioxidative activity of total phenols and antifungal activity in leaves and needles of the plants from the urban area and forest habitats were monitored. The obtained results indicate that urban areas induce changes in antioxidative metabolism in all examined species, but that the response is species specific. The most sensitive parameter that indicates different adaptation strategy of Pinus nigra, Picea omorika, Tilia cordata and Betula pendula to environment conditions in the urban area were peroxidase isoenzyme patterns. Less specific parameter was phenol content even though there are some indications for role of their antioxidative capacity in the adjustment to specific habitat. In addition, each species had different metabolic strategy to cope with the changes caused by the urban environment.

Specialist nectar-yeasts decline with urbanization in Berlin.

Nectar yeasts are common inhabitants of insect-pollinated flowers but factors determining their distribution are not well understood. We studied the influence of host identity, environmental factors related to pollution/urbanization, and the distance to a target beehive on local distribution of nectar yeasts within Robinia pseudoacacia L. and Tilia tomentosa Moench in Berlin, Germany. Nectar samples of six individuals per species were collected at seven sites in a 2 km radius from each target beehive and plated on YM-Agar to visualise the different morphotypes, which were then identified by sequencing a section of the 26S rDNA gene. Multivariate linear models were used to analyze the effects of all investigated factors on yeast occurrence per tree. Yeast distribution was mainly driven by host identity. The influence of the environmental factors (NO2, height of construction, soil sealing) strongly depended on the radius around the tree, similar to the distance of the sampled beehive. Incidence of specialist nectar-borne yeast species decreased with increasing pollution/urbanization index. Given that specialist yeast species gave way to generalist yeasts that have a reduced dependency on pollinators for between-flower dispersal, our results indicate that increased urbanization may restrict the movement of nectar-specialized yeasts, via limitations of pollinator foraging behavior.

Spatial and taxonomical overlap of fungi on phylloplanes and invasive alien ladybirds with fungal infections in tree crowns of urban green spaces.

Occurrence of entomopathogenic fungi on phylloplanes in Tilia × europaea crowns between 1 and 13 m was assessed in urban parks. Prevalence of fungal infections in ladybirds (Coleoptera: Coccinellidae) collected from Tilia × europaea was assessed to determine whether fungi found on phylloplanes also occurred as infections in ladybirds. Isaria spp. was most abundant on phylloplanes (mean colony forming units (CFU) per leaf ± SE, 0.33 ± 0.03) followed by Beauveria spp. (0.22 ± 0.02 CFU per leaf) and Lecanicillium spp. (0.19 ± 0.02 CFU per leaf). Densities of inoculum were higher in inner crowns and decreased with height, although Lecanicillium spp. peaked at 5-7 m. Upper phylloplane surfaces harboured higher densities of Isaria spp. and Beauveria spp. than lower surfaces, whereas Lecanicillium spp. was equally distributed. Most prevalent on ladybirds were Isaria spp. (20.6% Harmonia axyridis; 4.8% natives), Lecanicillium spp. (13.6% H. axyridis; 4.8% natives), with fewer Beauveria spp. infections (2.6% H. axyridis). Molecular identification revealed Beauveria bassiana, B. pseudobassiana, Isaria farinosa and Lecanicillium muscarium among isolates of both tree and ladybird origin. Tilia × europaea phylloplanes support a diverse assemblage of entomopathogenic fungal species with a different prevalence in coccinellids compared to their relative abundance in this habitat.

Ceratocystis tiliae sp. nov., a wound pathogen on Tilia americana.

Species in the North American clade (NAC) of the Ceratocystis fimbriata complex are mostly weak pathogens that infect native tree hosts through fresh wounds. Isolations from discolored tissue of wounded Tilia americana (basswood) in Iowa and Nebraska yielded a Ceratocystis species that was similar to but distinct from isolates of C. variospora from other hosts. Sequences of 28S rDNA showed that isolates from basswood did not differ from C. variospora, but there were minor differences in ITS rDNA sequences. The DNA sequences of a portion of the Cerato-platanin gene and TEF1α showed the basswood fungus to be a unique lineage. Cross inoculations in two experiments showed that the basswood isolates and C. variospora isolates from Quercus spp. were most aggressive to their respective hosts. Isolates from basswood grew slower and were less pigmented than C. variospora isolates from Quercus spp. The basswood fungus thus is distinguished from C. variospora based on phylogenetic analyses and phenotype and is herein described as C. tiliae sp. nov.

Characterization of fungal-degraded lime wood by X-ray diffraction and cross-polarization magic-angle-spinning 13C-nuclear magnetic resonance spectroscopy.

X-ray diffraction, scanning electron microscopy (SEM), and solid-state cross-polarization magic-angle-spinning (CP/MAS) (13)C-NMR spectroscopy were applied to determine changes over time in the morphology and crystallinity of lime wood (Tilia cordata Miller) generated by the soft-rot fungi. Wood samples were inoculated with Trichoderma viride Pers for various durations up to 84 days. Structural and morphological modifications were assessed by comparing the structural features of decayed lime wood samples with references. Significant morphology changes such as defibration or small cavities were clearly observed on the SEM micrographs of lime wood samples exposed to fungi. Following the deconvolution process of the diffraction patterns, the degree of crystallinity, apparent lateral crystallite size, the proportion of crystallite interior chains, and the cellulose fraction have been determined. It was found that all crystallographic data vary with the duration of exposure to fungi. The degree of crystallinity and cellulose fraction tend to decrease, whereas the apparent lateral crystallite size and the proportion of crystallite interior chains increase with prolonged biodegradation processes. The most relevant signals in CP/MAS (13)C-NMR spectra were assigned according to literature data. The differences observed were discussed in terms of lignin and cellulose composition: by fixing the lignin reference signal intensity, the cellulose and hemicelluloses moieties showed a relative decrease compared to the lignin signals in decayed wood.

Linden flower (Tilia spp.) as potential vehicle of Clostridium botulinum spores in the transmission of infant botulism / El té de tilo como vehículo potencial de esporas de Clostridium botulinum en la transmisión del botulismo infantil

Infant botulism is an intestinal toxemia caused principally by Clostridium botulinum. Since the infection occurs in the intestinal tract, numerous food products have been investigated for the presence of C. botulinum and its neurotoxins. In many countries, people use linden flower (Tilia spp) tea as a household remedy and give it to infants as a sedative. Therefore, to help provide a clear picture of this disease transmission, we investigated the presence of botulinum spores in linden flowers. In this study, we analyzed 100 samples of unwrapped linden flowers and 100 samples of linden flowers in tea bags to determine the prevalence and spore-load of C. botulinum. Results were analyzed by the Fisher test. We detected a prevalence of 3% of botulinum spores in the unwrapped linden flowers analyzed and a spore load of 30 spores per 100 grams. None of the industrialized linden flowers analyzed were contaminated with botulinum spores. C. botulinum type A was identified in two samples and type B in one sample. Linden flowers must be considered a potential vehicle of C. botulinum, and the ingestion of linden flower tea can represent a risk factor for infant botulism.

El botulismo del lactante es una toxiinfección causada, principalmente, por Clostridium botulinum. Debido a que esta infección ocurre en el tracto intestinal, la presencia de esta bacteria y sus neurotoxinas ha sido investigada en numerosos alimentos. En muchos países se utiliza el té de tilo (Tilia spp.) como sedante natural, el que se administra incluso a los lactantes. A fin de contribuir al esclarecimiento de la transmisión de esta enfermedad, se investigó la prevalencia y la carga de esporas botulínicas en esta hierba. Se analizaron 100 muestras de tilo comercializado a granel y 100 muestras de tilo industralizado en “saquitos”. Los resultados de prevalencia fueron analizados por el test de Fisher y la carga de esporas por la técnica del número más probable. Se halló una prevalencia de esporas de C. botulinum del 3% en el tilo comercializado a granel, con una carga de 30 esporas/100 g de hierba. En tanto, ninguna de las muestras en saquitos acusó la presencia del patógeno. Se identificaron tres cepas de C. botulinum, dos tipo A y una tipo B. En virtud de estos resultados, el tilo podría considerarse un potencial vehículo de esporas de C. botulinum y la administración de sus infusiones a menores y lactantes, un riesgo para la transmisión de la enfermedad.

Linden flower (Tilia spp.) as potential vehicle of Clostridium botulinum spores in the transmission of infant botulism.

Infant botulism is an intestinal toxemia caused principally by Clostridium botulinum. Since the infection occurs in the intestinal tract, numerous food products have been investigated for the presence of C. botulinum and its neurotoxins. In many countries, people use linden flower (Tilia spp) tea as a household remedy and give it to infants as a sedative. Therefore, to help provide a clear picture of this disease transmission, we investigated the presence of botulinum spores in linden flowers. In this study, we analyzed 100 samples of unwrapped linden flowers and 100 samples of linden flowers in tea bags to determine the prevalence and spore-load of C. botulinum. Results were analyzed by the Fisher test. We detected a prevalence of 3% of botulinum spores in the unwrapped linden flowers analyzed and a spore load of 30 spores per 100 grams. None of the industrialized linden flowers analyzed were contaminated with botulinum spores. C. botulinum type A was identified in two samples and type B in one sample. Linden flowers must be considered a potential vehicle of C. botulinum, and the ingestion of linden flower tea can represent a risk factor for infant botulism.

Role of sooty mold fungi in accumulation of fine-particle-associated PAHs and metals on deciduous leaves.

The focus of this research was on elucidation of the role of deciduous tree ecosystems in accumulation of fine-particle-associated polycyclic aromatic hydrocarbons (PAHs) and heavy metals on leaves of deciduous trees. The studied species were Tilia x euchlora (frequently infested by sooty mold fungi) and Pyrus calleryana (unaffected by sooty mold fungi). The selected species have similar leaf morphology and were exposed to identical environmental conditions. Intra-species comparison showed that moldy linden leaves accumulate significantly higher amounts of PAHs and metals than unaffected linden leaves. Inter-species comparison revealed that in the absence of sooty mold fungi, physico-chemical properties of epicuticular waxes, rather than the amounts of waxes, might play an important role in accumulation of particulate matter on leaves. The accumulation and/or degradation of a number of high-molecular-weight (HMW) PAHs on leaves was temperature dependent. The results show that the presence of sooty mold fungi on deciduous leaves alters either the accumulation modes and/or degradation pathways of PAHs on deciduous leaves.

Species diversity of polyporoid and corticioid fungi in northern hardwood forests with differing management histories.

Effects of forest management on fungal diversity were investigated by sampling fruit bodies of polyporoid and corticioid fungi in forest stands that have different management histories. Fruit bodies were sampled in 15 northern hardwood stands in northern Wisconsin and the upper peninsula of Michigan. Sampling was conducted in five old-growth stands, five uneven-age stands, three even-age unthinned stands and two even-age thinned stands. Plots 100 m x 60 m were established and 3000 m2 within each plot was sampled during the summers of 1996 and 1997. A total of 255 polyporoid and corticioid morphological species were identified, 46 (18%) of which could not be assigned to a described species. Species accumulation curves for sites and management classes differed from straight lines, although variability from year to year suggests that more than 2 y of sampling are needed to characterize annual variation. Mean species richness and diversity index values did not vary significantly by management class, although mean richness on large diameter wood (> or = 15 cm diam) varied with moderate significance. Richness values on small diameter debris varied significantly by year, indicating that a large part of year-to-year variability in total species richness is due to small diameter debris. Ten species had abundance levels that varied by management class. Two of these species. Changes in the diversity and species composition of the wood-inhabiting fungal community could have significant implications for the diversity, health and productivity of forest ecosystems.

[Interrelationships between yeast fungi and collembolans in soil].

The possibility of feeding on green and newly fallen leaves of the small-leaved lime Tilia cordata was studied for the collembolans Protaphorura armata and Vertagopus pseudocinereus. Young leaves grown under sterile conditions and almost free of yeast fungi were established to be toxic to the collembolan V. pseudocinereus: feeding on them led to the death of the animals. Leaves grown under natural conditions were nontoxic: when used by the collembolans as feed, they provided for collembolan growth and fecundity. Feeding preferences of the collembolans in relation to the yeasts attributed to different ecomorphs-epiphytes, litter saprophytes, pedobionts, and saccharobionts-were studied. Of the 24 yeast strains isolated from plant green parts, litter, and soil and assigned to eight species, no strain was revealed that was not used by the collembolans. However, certain yeast strains were preferable for the collembolans. The population of the V. pseudocinereus collembolans feeding on the yeast Rhodotorula glutinis (nss 31-4) exceeded that grown on Cryptococcus terricola (2044) 1.5-fold. Thus, the collembolans have feeding preferences in relation to yeast fungi, as was shown earlier for mycelial micromycetes. The possible mechanisms of the feeding preferences of the collembolans in relation to yeasts are discussed.

Fungal diversity during initial stages of leaf decomposition in a stream.

Maple, linden and oak leaves were immersed in a stream for 1-21 d. Cumulative mass loss, ergosterol content, and species richness of released aquatic hyphomycete conidia increased with time. Numbers and richness of attached conidia were highest on days 1 and 2. Denaturing gradient gel electrophoresis revealed up to seven fungal phylotypes on the leaves before their immersion in the stream and after one day of stream exposure. After 5 d of immersion the contribution of these terrestrial fungi decreased and that of aquatic hyphomycetes increased. The dominant phylotypes belonged to Anguillispora filiformis, Articulospora tetracladia and Flagellospora curvula, which also dominated the community of released spores. The molecular diversity was highest on day 2 and 3 on all substrates. This may be due to a few species of terrestrial fungi, later outcompeted by aquatic hyphomycetes, and to many different conidia of aquatic hyphomycetes, some of which may germinate but are unable to establish themselves and reproduce.

Two genetically related strains of Tuber borchii produce Tilia mycorrhizas with different morphological traits.

Two genetically related strains of Tuber borchii Vittad. (1BO and 43BO) produce mycorrhizas with Tilia platyphyllos Scop. with a different degree of efficiency. The aim of this work was to characterize the morphology of the fungal symbiotic structures in order to examine potential relationships between the anatomical traits of the mycorrhiza, the mycorrhizal capacities of the fungal strains and their effect on the host plants. Some morphological features of mantle hyphae (small size, intense staining, vacuolization, abundance of mitochondria) led to a mantle with morphological features that were isolate-specific. There were unexpected differences, at least under our experimental conditions: 1BO strain mantle cells were larger, less reactive to staining, more highly vacuolated and poorer in mitochondria than those of 43BO. These features were found throughout the mantle in 1BO, while the inner mantle hyphae of 43BO were significantly smaller and more intensely stained than the outer cells. In the 43BO strain there was a positive relation between these features and higher infectivity (evaluated as percentage of mycorrhizal tips) as well as a slightly more effective stimulation of plant growth. These observations suggest that genetically related truffle strains produce mycorrhizas with different morphologies, which may be related to a more efficient response of the host plant to inoculation.

Solid-phase microextraction gas chromatography/ion trap mass spectrometry and multistage mass spectrometry experiments in the characterization of germacrene D.

Germacrene D is a vegetable pheromone utilized in interactions among organisms belonging to different species. For the first time, using solid-phase microextraction/gas chromatography/ion trap mass spectrometry, the presence of this compound was detected in an in vitro mycorrhizal synthesis system where the mycelium of the ectomycorrhizal fungus Tuber borchii Vittad. interacts with the plant Tilia Americana L. From this symbiosis, a new structure, called ectomycorrhiza, is formed where the two symbionts exchange nutrients and metabolites. It seems that only after this interaction can the mycelium develop the fruitbody, commonly known as truffle. The results obtained allowed us to ascertain that germacrene D was synthesized by the plant exclusively in the presence of T. borchii. The originality of these data prompted us to hypothesize that this compound could be involved in the first step of ectomycorrhiza formation, as it is able to stimulate specific fungi receptors. In fact, plants release hundreds of secondary metabolites that are important in their interactions with other organisms.