Lifestyle-specific responses ofTrichodermaspp. in mycoparasitic confrontations and implications for biocontrol ofPopulusxcanescens

Antibiosis, biocontrol agent, chemotropism assay, ectomycorrhiza, host sensing, mycoparasitism, plant-pathogenic fungi, Trichoderma spp. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 3 1 Introduction Trichoderma species, belonging to the phylum Ascomycota, are naturally occurring in soil as well as on plant surfaces (Afzal et al., 2021; Oskiera et al., 2017). The genus of Trichoderma has been widely studied as a biocontrol agent (BCA) (Bonfante & Genre, 2010; Guzmán - Guzmán et al., 2019; Sharma et al., 2009; Sood et al., 2020; Stenberg et al., 2021). Biocontrol is based on several mechanisms, such as the antagonist ic activity against fungal plant pathogens, the proficiency to colonize plant tissues, the ability to induce systemic resistance in plants, as well as the adaptability to a wide range of environments (Liu et al., 2022; Manzar et al., 2022; Rush et al., 2021) . During the complex process of mycoparasitism, coherent mechanisms of antibiosis, competition for n utrients and space and direct inhibition by the release of fungal cell wall degrading enzymes (CWDE), such as chitinases, proteases, and β-glucanases, can effectively lead to inhibition and death of prey fungi (Manzar et al., 2022; Sharma et al., 2011; Thambugala et al., 2020; Viterbo & Horwitz, 2010) . The application of Trichoderma as BCA in disease management has already been shown to be effective against a broad range of foliar and root pathogens (Alfiky & Weisskopf, 2021; Benítez et al., 2005; Tyśkiewicz et al., 2022) . As a result, more than 60 % of the officially registered BCAs are based on different species of Trichoderma, with Trichoderma viride, Trichoderma virens, and Trichoderma harzianum being the most common ly used (Abbey et al., 2019; Nur & Noor, 2020; Rush et al., 2021). In addition to functioning as a bio -fungicide, numerous rhizosphere -competent Trichoderma spp. can form a close symbiosis with plant roots, producing soluble metabolites and volatile organic compounds (VOCs) with plant -performance stimulating activities conferring improved plant growth and induced resistance to abiotic stresses (Garnica-Vergara et al., 2016; Harman et al., 2004; Nawrocka & Małolepsza, 2013; Uniyal et al., 2018) . Therefore, the application of Trichoderma spp. as an environmentally friendly bio-fertilizer and BCA can minimize the amount of traditional fertilizers by improving nutrient and water acquisition, as well as reducing the amount of synthetic fungicides (Hermosa et al., 2013; Hyakumachi & Kubota, 2004; López-Bucio et al., 2015; Shoresh et al., 2010). Moreover, the described beneficial effects imply the potential economic impact through shortened plant production cycles and increased germination rates of seeds in nurseries (Aleandri et al., 2015; Grodnitskaya & Sorokin, 2006). was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 4 Hybrid poplars (Populus spp.) have gained significance due to their fast growth, making them a valuable feedstock for a wide range of wood and non -wood products with high economic importance, including bioenergy, building materials, and paper production (Polle & Douglas, 2010; Przybysz & Przybysz, 2013; Rubin, 2008). Populus spp. are naturally found in complex symbiotic interactions with beneficial microbes such as ectomycorrhizal fungi (ECM) (Karliński et al., 2010; Kwaśna et al., 2021; Luo et al., 2009; Plett & Martin, 2012; Schnitzler et al., 2010). However, monocultures of poplar hybrids in large-scale short rotation coppices (SRC) are also often susceptible to a wide range of soil-born and foliar fungal pathogens such as Armillaria root rot, Melampsora leaf rust, or leaf spot caused by Alternaria alternata (Hacquard et al., 2011; Ostry et al., 2014). Despite A. alternata being recognized as an airborn pathogen, its chlamydospores have been observed to persist both in soil and infested organic matter and was found on diseased roots of Vaccinium corymbosum and Taxus x media (Nadziakiewicz et al., 2018). Trichoderma spp. isolated from the rhizosphere of various trees have been shown to display a high antagonistic activity against common poplar pathogens, including A. alternata (Asef et al., 2008; Yu et al., 2022). However, the mycotrophic nature of Trichoderma bears the potential to have also negative effects on the local ECM population through competition for essential nutrients or growth inhibition by direct antagonism. While Trichoderma-based BCA might thus be a promising tool in SRCs to mitigate susceptibility to phytopathogenic fungi and improve biomass productivity, further research is needed to study the interaction between Trichoderma and plant-beneficial fungi in the rhizosphere, such as ECM partners, to evaluate potential risks of adverse effects on these non-target associations (Minchin et al., 2012), as these interactions play crucial roles in soil health and nutrient uptake (Frąc et al., 2018; Ma et al., 2008; Mayor et al., 2015). Existing literature reflects a degree of ambiguity regarding the ability of Trichoderma to differentiate between diverse fungal taxa, with important implications on the framework of the plant hol obiont theory (Hassani et al., 2018; Zilber -Rosenberg & Rosenberg, 2008) . Understanding wh ether Trichoderma can selectively interact with certain fungi while resisting/ignoring others would provide invaluable insights into the complex dynamics of plant- fungus interactions and ecosystem functioning. We therefore initiated a study to directly investigate the ability of Trichoderma spp. to distinguish between plant -beneficial ECM and plant-pathogenic fungi using representative species of each of these two major lifestyle groups. For this purpose, we selected two Trichoderma wild-type strains from the Harzianum was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 5 clade (one T. harzianum and one T. atrobrunneum strain), which we tested for their biofertilizer and biocontrol capacity in grey poplar (Populus x canescens, syn. P. alba x P. tremula). Confrontation scenarios were set up with the phytopathogens A. alternata and Fusarium graminearum in direct comparison with Laccaria bicolor and Hebeloma cylindrosporum as representative plant -mutualistic ECM. Moreover, to evaluate the physiological response of Trichoderma over longer distances and time wh ile having a two - directional choice of growth, we developed a novel olfactometer “race tube” -like system. Transcriptomic analysis was used to detect differences in the initiation of mycoparasitism - related programs during the different contact stages and better understand the interaction on a molecular level. 2 Materials and Methods Cultures and growth conditions The Trichoderma strain T. harzianum WM24a1 was obtained from the Austrian Institute of Technology GmbH (Monika Schmoll; Tulln, Austria), and T. atrobrunneum was isolated from a wood sample in 2018 (Bavaria, Germany). Both strains were identified previously on a molecular level following Cai & Druzhinina (2021) and tested in vitro for their biocontrol capacities (Stange et al., 2023). As potential preys the ECM basidiomycetes Laccaria bicolor isolate S238N (Institute National de la Recherche Agronomique, Nancy, France) and Hebeloma cylindrosporum (obtained from Technical Univ ersity Dresden, Germany) and the plant pathogens Fusarium graminearum PH-1 (obtained from University of Hamburg, Germany) and Alternaria alternata 22-2 (Phytopathology , Technical University of Munich) were used. Agar plugs from ECM fungi and F. graminearum, and spores from Trichoderma and A. alternata were routinely sub-cultured on potato dextrose agar and incubated at 21 °C and 75 % humidity in constant darkness. Populus x canescens INRA clone 717 1 -B4 was micropropagated routi nely in Schenk and Hildebrandt medium (SH medium, Schenk & Hildebrandt, 1972) as described by Behnke et al., 2007 and Müller, Volmer et al., 2013 and cultivated at 21°C, 75 % humidity, and 16 h photoperiod with 105 µmol -2s-1 (daylight white color 865). was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 6 Biofertilizer and biocontrol capacity in poplar Test plants (Supplementary Methods S1) were inoculated with five ml spore suspension of T. harzianum WM24a1 and T. atrobrunneum containing 10 6 spores ml-1 in sterile water , control plants were mock inoculated with sterile water. Directly afte r transfer and every two weeks plants were watered with ten ml of ¼ strength Long Ashton nutrient solution (Hewitt & Smith, 1975). The 5-week-old plants were inoculated again with five ml of a spore suspension containing 1 x 106 spores ml-1. After four days, one leaf per plant was wounded at f our sites with a sterile needle . For infection with A. alternata five µl of a spore solu tion containing 3 x 106 spores ml-1 was pipetted directly to the wounding site. For mock inoculation autoclaved water without spores was used and five replicates for each treatment were prepared. Pictures of the leaves were taken after five days and the total infection area per leaf was measured using ImageJ V1.53e software (Wayne Rasby, National Institute of Health, USA, http://imageJ.nih.gov/ij). Plant height, leaf number and shoot and root fresh weight were assessed for a subset of plants (n = 7) after 6 weeks. Dry weight was determined after 48 h at 50°C. Antagonistic activity of Trichoderma For in vitro antagonism assays in dual culture, the second fungal partners H. cylindrosporum, L. bicolor, F. graminearum, or A. alternata were inoculated on solid Modified Melin-Norkrans synthetic medium (MMN) (Müller; Volmer et al. 2013) in non-split and split Petri dishes (9 cm diameter) (Guo et al., 2019) and the ECM were incubated for two and the pathogens one weeks, respectively. Trichoderma strains were inoculated on the other side of the plate as spores and after three days photos were taken with a Nikon camera (Nikon, Tokyo, Japan) The colony area (cm 2) of fungal mycelium in media contact (MC) and air contact (AC) was measured using ImageJ. As a control, each fungus wa s grown alone. The inhibitory effect was calculated by using the following formula (Raut et al., 2014): Change in colony size compared to control (%) = D1 − D2 D1 × 100 With D1 = colony area of control condition and D2 = colony area of test condition was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 7 Physiological response of Trichoderma using race tube system The olfactometer “race tube”-like system (Supplementary Fig. S2, Methods S2) was composed of two 220 ml sample cups (391 -0023, VWR, Darmstadt, Germany) and a 50 ml serological pipette (612-3696, VWR, Darmstadt, Germany). L. bicolor and H. cylindrosporum as well as F. graminearum and A. alternata were inoculated into the right tube two and one week before Trichoderma was inoculated with spore solution into the middle of the race tube. For AC condition the second fungus was inoculated into a small petri dish and placed into the test cup. Both cups were sealed with a gas -permeable membrane (BR701364, Sigma , St. Louis, USA ). The left tube remained uninoculated, serving as a control tube. Growth of Trichoderma was monitored at 48 h, 72 h, 96 h, 120 h, and 144 h , respectively, after inoculation by marking the hyphal growth on the corresponding section of the serological pipette. As a control, Trichoderma was challenged with itself. The direction of hyphal growth of Trichoderma was determined by the percentage of total growth between the described time points and ∆ % of growth was calculated with the following formula: % of total growth towards partner − % of total growth towards control = ∆ growth direction [%] Transcriptomic analysis of confrontations with phytopathogens and ectomycorrhiza To determine whether the presence of a plant-beneficial and a plant-pathogenic prey affects the expression of mycoparasitism-related genes during different degrees of contact with ECM and plant -pathogens, an RNA -Seq experiment was performed. Confrontation scenarios where the same as for testing antagonistic activity, consistent with modified MMN media overlaid with wet autoclaved cellophane membrane (Natureflex 32g m-2, HERA, Schotten, Germany) to enable biomass harvest without agar residues. The biomass was harvested three and six days after Trichoderma inoculation with a sterile spatula and subjected to RNA extraction ( Supplementary Methods S3). Qualified RNA was subjected to cD NA library preparation using the Illumina stranded mRNA-Kit (Illumina, San Diego, USA) quantified and qualified (DNA High Sensitivity assay, Aligent Technologies, Santa Clara, USA) by the chair of Animal Physiology and Immunology at TUM. Sequencing of barc oded libraries was done at IMGM laboratories (Martinsried, Germany) with NovaSeq 6000 for 100 bp single-end reads to a depth of 18 million reads per sample. H. cylindrosporum could not be included in this transcriptomic analysis, since it was not available at that time. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 8 Bioinformatic analysis Sequencing data was processed using nf-core/rna-seq v3.12.0 workflow (Ewels et al., 2020) and executed with Nextflow v23.10.0 (Di Tommaso et al., 2017) . The reads were trimmed using Cutadapt (v4.8) (Martin, 2011) to remove poly -A tails, adaptor sequence contaminations and low -quality bases and subsequently aligned to the concatenated

genomes with STAR (Dobin et al., 2013). Gene-level read counts were determined using Salmon (Patro et al., 2017) and subjected to downstream analysis. Differential gene expression analysis was conducted using the DESeq2 R package (v1.16.1) to normalize the libraries based on the geometric mean of the read counts and then calculate the log2fold change (LFC) between the experimental test conditions and the control condition (Love et al., 2014). Genes were identified to be differentially expressed (DEGs) with adjusted p-value (FDR) 0) were submitted to FungiFun 2.2.8 (Priebe et al., 2015) with T. harzianum CBS 226.95 and L. bicolor S238N-H82 / ATCC MYA -4686 as

species, to assign functional annotations of DEGs and conduct enrichment analyses. DEGs were classified based on Functional Catalogue (FunCat) (Ruepp et al., 2004), Gene Ontology (GO) (Harris et al., 2004) , and Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa & Goto, 2000) and tested for enrichment using FungiFun 2.2.8 BETA (Priebe et al., 2015) . The presence of signal peptides in DEGs was predicted with SignalP 5.0 (Almagro Armenteros et al., 2019) . Self-organizing tree algorithm (SOTA) was used to cluster common DEGs based on expression patterns between the different test conditions using Pearson’s correlation and MeV v4.4.1 (Howe et al., 2011). Statistical analysis and visualization The obtained data was processed for statistical data analysis with Origin Pro (version 2021, OriginLab Corporation, Northampton, USA). Data was tested for normal distribution using the Shapiro-Wilk test, followed by Levene’s test for variance homogeneity. One-way ANOVA was performed combined with Bonferroni test for pairwise comparison. For plant data, one -way ANOVA was applied combined with Fisher’s least significant difference (LSD) test. Figures were created and combined with BioRender.com (TUM institutional license WN26OFV8MS). was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 9 3 Results Trichoderma spp. are displaying bio-fertilizer and biocontrol capacity in poplar When considering Trichoderma strains as a BCA for poplar plantations, the potential to induce effective plant systemic resistance may vary between different strains used. I t is therefore important to exclude potential negative effects by eva luating the interaction of the biocontrol strain and the plant. The biofertilizer capacity was evaluated by comparing plant height, leaf number, shoot and root fresh and dry weight to the untreated control plants (Fig. 1). The chosen strains significantly increased plant height, average leaf number (from 7.2 (+/- 1.6) to 8.4 (+/- 0.9) with T. harzianum treatment and 9.4 ( +/- 1.2) for T. atrobrunneum treatment) (Fig. 1a,b), as well as shoot and root fresh weight (Fig. 1c,d). To evaluate the potential biocontrol capacity of both strains, one leaf of each poplar-plant was injured and infected with spores of A. alternata. Both Trichoderma strains significantly decreased signs of infection in leaves (Fig. 1g) reflecting a positive influence on the induced systemi c resistance in P. x canescens. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 10 Fig. 1: Evaluation of biofertilizer and biocontrol capacity of T. harzianum WM24a1 and T. atrobrunneum in Populus x canescens. Plant height (a), leaf number (b), shoot fresh and dry weight (c,e) and root fresh and dry weight (d,f) were assessed after six weeks of cultivation (n = 7). A subset of plants were inoculated again with 5 ml spore solutions containing 106 spores ml-1. After 4 days one leaf per plant was injured with a sterile needle and infected with A. alternata spore solution (n = 5). Infection area in mm2 (g). Significances were determined by one-way ANOVA and LSD, p < 0.05. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 11 Differences in antagonistic activity of Trichoderma towards plant-beneficial and plant- pathogenic fungi To examine the physiological response and potential inhibitory effect of Trichoderma species towards plant -pathogenic and plant -beneficial fungi , we initially set up dual confrontation assays with F. graminearum , A. alternata, L. bicolor and H. cylindrosporum , in different degrees of contact in standard Petri dish systems (Supplementary Fig. S1). A clear inhibitory effect of both Trichoderma strains towards all confrontation partners was observed three days after inoculation with Trichoderma, albeit differing in severity depending on the part ner and being generally stronger during conditions that allowed media contact (MC), compared to the situation in a split -plate that allowed only contact through the headspace (air contact; AC). Also, T. harzianum showed a stronger inhibitory effect on both pathogens during MC compared to AC, whereas T. atrobrunneum only showed stronger inhibitory effect s during MC towards A. alternata (Fig. 2c,d). The inhibitory effect of Trichoderma towards the ECM was stronger towards H. cylindrosporum compared to L. bicolor for both strains (Fig. 2a,b). On the contrary, the presence of H. cylindrosporum and L. bicolor was found to inhibit the growth of both Trichoderma strains (between 4% and 47%) (Fig. 2b), and this effect was more pronounced, albeit not significantly, in the AC conditions and somewhat more robust for L. bicolor than for H. cylindrosporum (Fig. 2a,b). At the same time, the presence of both pathogens led to an enhanced colony diameter of Trichoderma, indicated by negative inhibition values (Fig. 2c,d). In T. atrobrunneum, this effect ranged between 2% and 10% and in T. harzianum between 11% and 29% compared to the Trichoderma-only control. Intriguingly, while this increase was stronger during AC confrontation with F. graminearum compared to MC confrontation, no significant differences could be detected between AC and MC confrontation with A. alternata. Overall, these data show that while the ECM fungi have a robust repelling effect on Trichoderma spp., they seem to be attracted to plant-pathogenic fungi. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 12 Fig. 2: Growth inhibition during co-cultivation of Trichoderma spp. with the two ectomycorrhizal fungi H. cylindrosporum (a) and L. bicolor (b) and the two plant-pathogenic fungi F. graminearum (c) and A. alternata (d). Growth inhibition of each fungus during co-cultivations was calculated by comparing the colony area with single controls for MC and AC three days after inoculation with Trichoderma. Significances were determined for each contact stage separately with Student’s t-test, p < 0.05, n = 3. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 13 A novel olfactometer “race tube”-like system to quantify the directional growth response to fungal partners To overcome the limitations of more traditional plate confrontation assay s, we developed a novel olfactometer “race-tube”-like system. Differing from the Petri dish system, this experimental set -up allows for a two -way choice of growth direction and therefore an observation of the directed growth of Trichoderma in presence of a second fungus (Fig. 3a). Furthermore, the growth direction can be evaluated and quantified over much longer distances and time and therefore in a much more reliable fashion. Self -confrontation of both Trichoderma strains led to Δ growth direction values near 0, indicating an equal growth towards both directions (Fig. 3b-e). Using the new system, we could confirm the overall effects observed in the plate -based system. However, it became obvious that both Trichoderma strains were not simply inhibited by the ECM fungi, but indeed chose to grow away from them, visible by stronger growth in the opposite direction, leading to negative Δ growth direction values. This effect was stronger for T. harzianum during AC compared to MC (Fig. 3b,c), whereas it was the other way around for T. atrobrunneum (Fig. 3d). The situation was found to be completely different in presence of phytopathogens, and both Trichoderma strains displayed a clear growth preference towards those fungi, as indicated by positive Δ growth direction values (Fig. 3b-e). In T. harzianum this effect was stronger during the first 72 h in AC compared to MC, shifting to more pronounced effects in MC compared to AC after 96 h (Fig. 3b,c). Interestingly, in both Trichoderma strains the directed growth towards the pathogens in AC confrontation decreased slightly after 96 h, and in MC confr ontation after 120 h. Overall, the physiological responses indicated a negative chemotropism in presence of ECM and a positive chemotropism in presence of phytopathogenic fungi taking effect already at comparably long distances. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 14 Fig. 3: Experimental setup of olfactometer “race tube”-like system and the confrontations with fungal partner during media (MC) and air contact (AC) (a). Physiological response of T. harzianum WM24a1 (a,c) and T. atrobrunneum (d,e) in the presence of H. cylindrosporum (grey), L bicolor (blue), F. graminearum (red) and A. alternata (orange). The direction of growth was determined as ∆ growth direction (%) between the different time points. As a control, Trichoderma was challenged with itself (green). Significances were determined with Student’s t-test compared to the self -confrontation, p < 0.05, n = 5. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 15 Distinct patterns in Trichoderma global gene expression To identify differentially expressed genes (DEGs) related to the strongly differing reaction of Trichoderma to ECM and phytopathogenic fungi, another series of plate-based confrontations was conducted with T. harzianum on one side and L. bicolor, F. graminearum, or A. alternata on the other. The plate system was used in this experiment , since it allowed harvesting biomass directly from the zone of interaction (Fig. 4a). Samples were taken three days after inoculation before any kind of physical contact (MC) and six days after inoculation when a direct hyphal interaction was established (DC). Compared to the concatenated reference genomes of T. harzianum CBS 226.95 and Laccaria bicolor S238N-H82, the alignment rate ranged from 81.2% to 97.7% , respectively. Principal component analysis (PCA) revealed cluster formation of the c onfrontations with the pathogens away from the Trichoderma-only control, indicating similarly changed expression patterns, while confrontation with L. bicolor clustered much closer to the controls, indicating more limited changes (Fig. 4b). At DC, the pathogen interactions separated into individual clusters, while the confrontation with L. bicolor still clustered with the control (Fig. 4c). This differential clustering highlights the similarities between confrontation with L. bicolor and the control condition and points to distinct expression patterns during the confront ation with the pathogens developing after three days. Distinct patterns across the confrontations with the ECM and the pathogens during MC emerged also when looking at the gene expression more closely (volcano plots; Supplementary Fig. S3). The confrontation with L. bicolor was characterized by small LFC values compared to the control, representing minimal changes in gene expression. To nevertheless ensure not to lose potentially relevant genes in the downstream analysis, we employed a threshold of adjusted FDR < 0.01 without an additional LFC threshold. Especially during the MC stage without physical contact, we expected the potential signaling mechanisms not to display extreme fold -changes, which should nevertheless be significant. This approach allowed to capture the nuanced variations in gene e xpression during confrontation with the ECM. Conversely, in confrontation with the pathogens during the MC, the volcano plots illustrate a broader dispersion of points with higher LFC values, indicating a notable and pronounced alteration in gene expression. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 16 Fig. 4: Transcriptomic analysis of fungal confrontations. Experimental setup alongside with depicting areas of biomass harvest from the zone of interaction (a). PCA plots illustrate differential gene expression in T. harzianum after three (MC) and six days (DC) (b,c) and in L. bicolor (d,e) based on the top 500 differentially expressed genes among all conditions. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 17 Unique genetic response of T. harzianum towards L. bicolor Overall, the interaction of T. harzianum and L. bicolor during MC (three days) led to the up- and downregulation of only 67 and 56 DEGs in T. harzianum, respectively (Fig. 5a). This observation aligns with the previous observations . The changes in the transcriptome of T. harzianum were small compared to the distinct and pronounced cha nges during confrontation with F. graminearum and A. alternata, which led to 614 and 1,072 upregulated DEGs and 1,262 and 893 downregula ted DEGs, respectively. During DC (day six) 366 and 175 genes were significantly up - and downregulated in T. harzianum during interaction with L. bicolor, whereas the presence of both pathogens led to much more DEGs (2,366 and 1,692 upregulated DEGs during confrontation with A. alternata and F. graminearum, respectively) (Fig. 5a). After three days of confrontation with the two pathogens, 767 and 483 DEGs were commonly up- and downregulated in T. harzianum (Fig. 5c,d), while during confrontation with L. bicolor (TL3), 65 and 46 were uniquely up - and downregulated, respectively. During overgrowth stage (DC, six days), confrontation with pathogens up - and downregulated 873 and 831 shared DEGs in T. harzianum, while only 75 and 58 DEGs were common also in the confrontation with L. bicolor (Fig. 5e,f). was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 18 Fig. 5: Number of DEGs (FDR < 0.01) that were up - or downregulated in the co nfrontation of T. harzianum with L. bicolor (TL), A. alternata (TA), and F. graminearum (TF) compared to the control condition after three days (MC) (TL3, TA3, TF3) and six days (DC) (TL6, TA6, TF6) (a). Number of DEGs (FDR < 0.01) that were up- or downregulated in the confrontation of L. bicolor with T. harzianum (LT) after three and six days (LT3 and LT6, respectively) (b). Venn diagrams showing common and unique DEGs among the three different confrontations of T. harzianum with L. bicolor (TL), A. alternata (TA), and F. graminearum (TF) after three days (c,d) and after six days (e,f). was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 19 During MC (three days) , at least 13 genes from among the common and most upregulated 30 DEGs in confrontation with A. alternata (TA3) and F. graminearum (TF3) are known to play a crucial role in the process of mycoparasitism, clearly showing induction of related gene cascades already long before direct hyphal contact. On the contrary, during confrontation with L. bicolor (TL3) those mycoparasitism -related genes rather showed a downregulation (Table 1). Interestingly, a terpene synthase (M431DRAFT_113113; LFC -0.9) was significantly downregulated as well . Furthermore, two short and uncharacterized signal peptide-containing proteins (M431DRAFT_69921, M431DRAFT_129453) were signi ficantly induced. Table 1: Genes in T. harzianum with known or predicted functions in the process of mycoparasitism that were found to be upregulated during the “sensing -phase” in presence of A. alternata (TA3) and F. graminearum (TF3), but not L. bicolor (TL3), at 3 days post inoculation. Predicted protein names were retrieved from UniProt and log2 fold-changes (log2FC) of all three confrontations are displayed and highlighted red for log2FC log2FC > 1 and green for log2FC > 1. Genes which were removed due to low counts show log2FC of NA. DEGs containing a signal -peptide predicted by SignalP are marked with a †. Predicted protein name Gene ID log2FC TA3 log2FC TF3 log2FC TL3 Assigned GO terms glucan endo-1,3- beta-D- glucosidase† M431DRAFT_479664 2.84 4.50 0.13 beta-glucosidase activity [GO:0008422]; polysaccharide catabolic process [GO:0000272] Carbohydrate- binding module family 24 protein† M431DRAFT_525334 1.87 3.56 - 0.53 glucan endo-1,3- alpha-glucosidase activity [GO:0051118] Oligopeptide transporter M431DRAFT_529850 2.45 3.51 - 0.67 oligopeptide transmembrane transporter activity [GO:0035673]; membrane [GO:0016020] Peptidase S1 domain-containing protein† M431DRAFT_526221 2.13 3.37 - 0.62 serine-type endopeptidase activity [GO:0004252]; proteolysis [GO:0006508] Glycoside hydrolase family 18 protein† M431DRAFT_509593 2.48 3.30 - 0.23 hydrolase activity [GO:0016787]; carbohydrate metabolic process [GO:0005975] was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 20 Carbohydrate- binding module family 13 protein M431DRAFT_9084 1.73 2.17 - 0.35 carbohydrate metabolic process [GO:0005975] Glycoside hydrolase family 3 protein, β- glucosidase† M431DRAFT_504078 1.65 1.97 - 0.51 hydrolase activity, hydrolyzing O- glycosyl compounds [GO:0004553]; polysaccharide catabolic process [GO:0000272] Chitinase† M431DRAFT_505895 1.46 1.95 - 0.32 chitin binding [GO:0008061]; chitinase activity [GO:0004568]; chitin catabolic process [GO:0006032]; polysaccharide catabolic process [GO:0000272] Chitinase† M431DRAFT_500888 2.30 1.89 NA cellulose binding [GO:0030248]; chitinase activity [GO:0004568]; chitin catabolic process [GO:0006032]; polysaccharide catabolic process [GO:0000272] Amino acid permease/ SLC12A domain-containing protein M431DRAFT_92558 1.07 1.55 - 0.44 membrane [GO:0016020]; amino acid transport [GO:0006865]; transmembrane transport [GO:0055085] Major facilitator superfamily (MFS) profile domain- containing protein M431DRAFT_101977 1.65 1.52 0.84 transmembrane transporter activity [GO:0022857]; membrane [GO:0016020] Glycosyl- transferase family 32 protein M431DRAFT_238777 0.94 1.49 - 0.17 alpha-1,6- mannosyltransferase activity [GO:0000009]; carbohydrate derivative metabolic process [GO:1901135] Chitinase† M431DRAFT_517960 0.74 1.37 0.05 chitin binding [GO:0008061]; chitinase activity [GO:0004568]; chitin catabolic process [GO:0006032]; polysaccharide catabolic process [GO:0000272] was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 21 Gene ontology (GO) analysis of upregulated genes showed clear enrichment (exact Fisher test, adj. p-value > 0.05) of terms involved in primary metabolic activity, such as “ribosomes”, “translation” and “carbohydrate metabolic process” (Fig. 6a). This aligns with the phenotypic observations of increased colony area when T. harzianum was confronted with plant pathogens. Furthermore, GO terms of “cellulose binding”, “chitinase activity” and “hydrolase activity” were significantly enriched, corroborating that Trichoderma is able to sense potential prey already before direct hyphal contact. Conversely, GO enrichment analysis of upregulated DEGs in interaction with L. bicolor revealed a much more limited outcome with membrane-related annotation being the only enriched category (Fig. 6b). was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 22 Fig. 6: Gene ontology enrichment analysis of common and unique upregulated DEGs in T. harzianum during the interaction wit h A. alternata (TA3, TA6) and F. graminearum (TF3, TF6) after three days (MC) (a) and six days (DC) (c). GO enrichment analysis of DEGs uniquely upregulated in T. harzianum in presence of L. bicolor after three days (TL3) (b) and six days (TL6) (d). GO terms were assumed to be significantly enriched with adjusted p-value < 0.05. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 23 Intriguingly, we identified 57 genes that were differentially expressed in all three test conditions during MC (three days), but which nevertheless show distinct expression patterns, according to the lifestyles . Expression profiles of those common DEGs by SOTA method using Pearson’s correlation revealed two distinct clusters (Fig. 7, Supplementary Fig. S4, Table S2): 42 genes (cluster 1 ) showed a significant induction in T. harzianum during confrontation with L. bicolor and significant repression during confron tation with the pathogens. The remaining 15 DEGs (cluster 2 ), s howed an opposite regulation, being significantly upregulated during the confrontation with the pathogens and down regulated during confrontation with L. bicolor. Several DEGs in cluster 1 are assigned to GO terms of “membrane” and associated with transport activities , as seen above. Interestingly, also a small secreted protein (M431DRAFT_96469; signal peptide likelihood: 0.99) was significantly induced in confrontation with L. bicolor with an LFC of 0.83 (FDR 0.001) and downregulated in presence of A. alternata and F. graminearum with LFC of -0.99 and -1.15 (FDR 2.75E-8 and 1. 45E-6), respectively. Cluster 2 contains se veral DEGs annotated with GO terms of “hydrolase activity” and a peptidase A4 family protein upregulated with LFCs of 0.80 and 1.01 (FDR 2.96E -11 and 1.23E-7) in confrontation with A. alternata and F. graminearum , respectively, and downregulated in confrontation with L. bicolor (LFC of -1.19; FDR 3.7E-14). GO-term enrichment analysis of T. harzianum genes upregulated at day six of contact with both pathogens identified “membrane” -related categories, “transport”, as well as “chitin catabolic processes”, as to be expected during mycoparas itism (Fig. 6c). During interaction with the ECM, significantly enriched GO terms were pred ominantly associated with (extracellular) carbohydrate metabolism and cell wall organization (Fig. 6d). Overall, many pr ocesses related to mycoparasitism were upregulated already in the early stage of interaction with the two pathogens, while the presence of L. bicolor did not lead to any strong induction or repression of specific genes. Also during DC, only contact with the plant pathogens led to clearly recognizable antagonistic gene expression patterns. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 24 Fig. 7: Heatmap of the 57 common DEGs in T. harzianum after three days during confrontation with L. bicolor (TL3), A. alternata (TA3) and F. graminearum (TF3) shown as log2 fold change (normalized to single control of T. harzianum TC3) and clustered based on expression using SOTA. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 25 Transcriptomic alterations in L. bicolor in confrontation with T. harzianum To identify ECM genes involved in the interaction with Trichoderma, we next i nvestigated transcriptomic changes in L. bicolor during MC and DC with T. harzianum. The LFC values were similarly small as in T. harzianum during interaction with L. bicolor (Supplementary Fig. S3). PCA analysis of the L. bicolor transcriptome revealed ve ry similar expression patterns like in the single fungus controls at 3 days (MC) and only clear distinct clustering during DC (six days; Fig. 4d,e). During MC, 117 and 329 DEGs were up - and downregulated, respectively, in L. bicolor in confrontation with T. harzianum (Fig. 5b). Enrichment analysis of upregulated DEGs in L. bicolor after three days revealed enrichment ( p-adj. < 0.05) of only one GO term, “DNA binding transcription factor activity ” (GO:0003700), while no pathways in FunCat or KEGG were significantly enriched (Supplementary Table S4). However, several of the upregulated DEGs were annotated with FunCat main categories “Transcription”, “Metabolism”, “Protein with binding function or cofactor requirement”, and “Cellular communication / signal transduction”. This includes o ne Nrg1 -like Zn -finger transcription factor (LACBIDRAFT_296037) being significantly induced in presence of T. harzianum, as well as two SNF2 family DNA-dependent ATPases (LACBIDRAFT_301027, LACBIDRAFT_396054). Notably, three upreg ulated DEGs ( LACBIDRAFT_240638, LACBIDRAFT_246709, LACBIDRAFT_248257) were linked to “G-protein coupled receptor signalling, cellular communication / signal transduction mechanism” pathways, indicating the initiation of signaling cascades in L. bicolor as potential response to signaling molecules derived by T. harzianum. Moreover, several of the 117 upregulated DEGs were assigned to subcategories of the KEGG main pathways “Metabolism”, such as “Biosynthesis of secondary metabolites”, “Steroid biosynthesis”, “alpha-Linolenic acid metabolism”, “Terpenoid backbone biosynthesis”, and “Fatty acid metabolism”, potentially representing an activation of secondary metabolite-based communication. Interestingly, the most upregulated gene, LACBIDRAFT_304386 (LFC 0.86), was predicted to be a signal peptide-containing protein (likelihood of 0.99). The third most upregulated DEG was an oligopeptide transporter (LACBIDRAFT_302225 ; LFC 0.71), suggesting increased fluxes of metabolites. A slightly upregulated signal peptide -containing (likelihood 0.94) tripeptidyl-peptidase II (LACBIDRAFT_191088) might indicate a very moderate triggering of was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 26 some defense mechanisms. Moreover, a plasma membrane fusion protein (LACBIDRAFT_231982), part of the fusion machinery and involved in stabi lizing the plasma membrane (“mating projection tip” GO:0043332; “plasma membrane” GO:0005886) was increased by 39%, which could be indicating a reaction of cell wall and membrane remodeling in response to secreted enzymes or effector proteins by T. harzianum. The DC condition led to a stronger response, with 2314 and 1743 up - and downregulated DEGs (Fig. 5b), respectively. Upregulated DEGs revealed enriched GO terms of “oxidation - reduction process” (GO:0055114), “oxireductase activity” (GO:0016491), “regu lation of transcription” (GO:0006355), and “metal ion binding” (GO:0046872) (Fig. 8a). From KEGG main category “metabolism”, the pathways of “oxidative phosphorylation”, “citrate cycle (TCA cycle)”, “valine, leucine and isoleucine degradation”, “pyruvate m etabolism” and “carbon metabolism” were significantly enriched, and FunCat categories such as “cellular transport, transport facilitation and transport routes” (38.4%) “metabolism” (18.4%), “energy” (15.7%), “protein with binding function of cofactor requi rement” (12.8%), “cell rescue, defense and virulence” (11.4%), and “interaction with the environment” (1.8%) (Fig. 8b). Among the transport-related categories , FunCat descriptions of “endoycytosis”, “drug/toxin transport”, and “ABC transporters” (Fig. 8c) also indicate defense mechanisms. One of those assigned DEGs was a glutathione transferase (LACBIDRAFT_188517; LFC 5), with known function in detoxification, as well as a multidrug resistance -associated (MDR) ABC transporter (LACBIDRAFT_318236, LFC 2), and a pleiotrophic drug resistance (PDR) ABC transporter (LACBIDRAFT_314719, LFC 2.5). Furthermore, several Mycorrhiza -induced Small Secreted Proteins (MiSSPs) such as MISSP6.4 (LACBIDRAFT_316998 ; LFC 5.57 ), MISSP16.2 (LACBIDRAFT_333197 ; LFC 1.9), and MISSP2 2.4 (LACBIDRAFT_303456 ; LFC 0.48 ) were significantly upregulated during “overgrowth -phase”. MiSSPs are known to play various roles in plant -fungus interactions, functioning as signaling molecules or effectors. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 27 Fig. 8: Gene ontology enrichment analysis of upregulated DEGs in L. bicolor in presence of T. harzianum after six days of co -cultivation (DC) (a). FunCat enrichment analysis of upregulated DEGs in L. bicolor during DC (six days) with T. harzianum . Significant enriched FunCat main categories (b) and enriched subcategories of “Cellular transport, transport facilitation and transport routes” (c). GO terms and FunCat categories were assumed to be significantly enriched with adjusted p-value < 0.05. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 28 4 Discussion Fungi engage in diverse interactions with their environment including nearby organisms. The fungi used in this study ( Trichoderma spp., Laccaria spp., Hebeloma spp., Fusarium spp., and Alternaria spp.) can all be found in the soil and rhizosphere and occupy similar environmental niches, while nevertheless having different lifestyles (Hagn et al., 2003). The interactions between two fungal species can manifest as either antagonistic, parasitic, neutral, or synergistic, each yielding distinct outcomes , both for the fungi as well as for potentially involved further interaction partners, such as plants (Venturi & Keel, 2016; Whipps, 2001). One can assume that it would be in the interest of the plants to maximize the number of plant -beneficial microorganisms in their rhizosphere. Trichoderma spp. are particularly interesting in this regard, since mycotrophy is thought to be an ancient trait of the entire genus, while many have been shown to work as plant fertilizers (Druzhinina et al., 2011). However, whether the application of Trichoderma comes at the expense of overall fungal biodiversity, including plant -beneficial fungi, or is somewhat selective, is controversial so far. A risk assessment when considering Trichoderma spp. as biocontrol measure was therefore suggested already early on, requiring to identify the effect on the target (pathogen) population while evaluating negative effects on non -target (native and plant -beneficial) fungal species, which would have detrimental consequences for the host plant (Brimner & Boland, 2003). Trichoderma viride and T. polysporum, for example, can have a significant impact on ECM, since these species were found to strongly inhibit mycorrhization of black spruce seedlings by L. bicolor (Summerbell, 1987) . Conversely, the application of the Trichoderma bio- inoculant ArborGuard™ on Pinus radiata seedlings did not adversely affect the ECM colonization in a nursery system, but did also not promote seedling growth (Minchin et al., 2012). Another study investigated the impact of T. virens on pre-mycorrhized Pinus sylvestris roots. Intriguingly, while a decreased spore germination of Trichoderma was monitored in the rhizosphere, indicating the presence of dampening plant - or ECM -derived processes, the overall plant viability was influenced positively (Werner et al., 2002). The described inhibitory effect of ECM towards Trichoderma spp. was observed across various in vitro and in planta experimental setups (e.g. Guo et al., 2019; Minchin et al., 2012; Summerbell, 1987; Werner et al., 2002 ). A similar inhibitory effect of the related ECM Laccaria laccata towards Trichoderma virens in co-culture was already described by Werner et al. (2002). Moreover, Summerbell (1987) reported the absence of typical hyphal structures of T. viride towards was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 29 L. bicolor during their interaction, and Zadworny et al. (2007) also described a decreased colony area of T. virens and T. harzianum during co-culture, as well as altered microtubular cytoskeleton structures in the interaction zone of both fungi, with more pronounced effects in the saprotrophic strains, indicating a stress response. Likely, the variable outcomes of interaction studies are strongly influenced by the experimental setup, such as space and nu trients, which can unintentionally force the interactions into a specific direction. Our results now demonstrate that in the presence of enough space, Trichoderma spp. prefer to grow away from ECMs and do not initiate mycoparasitic programs (either at the physiological or molecular level ), that occur in the presence of pathogens long before direct physical contact. These results are in support of a hypothesis arguing in favor of regulatory processes that have evolved to maximize the number of plant-beneficial fungi in the rhizosphere. Trichoderma are attracted by plant-pathogens and avoid ECM The dual confrontation assays performed in this study revealed intriguing insights into the response of Trichoderma towards fungi of different lifestyles. Co -culture s ystems are representing a forced competition of two organisms to decide on the fate of the limited resources in confined spaces. Notably, Trichoderma strains displayed varying inhibitory effects against F. graminearum , A. alternata , H. cylindrosporum, and L. bicolor during different contact conditions. T. harzianum exhibited stronger inhibitory effects towards F. graminearum and A. alternata , especially during MC, suggesting its robust potential to produce diffusible, non -volatile secondary metabolites havi ng a significant effect on the growth of plant -pathogenic fungi (Guo et al., 2019; Küçük & Kivanç; Qualhato et al., 2013; Sood et al., 2020). Fungal interactions are facilitated through the exchange of signaling molecules, which are released and secreted by one fungal species and subsequently perceiv ed by receptors present in the other participating species (Carreras-Villaseñor et al., 2012; Raut et al., 2014; Zeilinger & Atanasova, 2020). Fungal VOCs are organic chemicals with low molecular weight that originate from metabolic processes within the fungus, evaporate easily at moderate temperature (Macías-Rodríguez et al., 2015) and participate in the communication between fungal species (Guo et al., 2019; Guo et al., 2020; Weikl et al., 2016; Wonglom et al., 2020). Several studies suggest that fungal VOC profiles are similar for fungi with comparable lifestyles (El Jaddaoui et al., 2023; Farh & Jeon, 2020; Guo et al., 2020; Guo et al., 2021; was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 30 Müller, Faubert et al., 2013; Razo -Belmán et al., 2023) and also Trichoderma species have been demonstrated to produce several volatiles (many being sesquiterpenes), as well as receive, and respond to VOCs (Guo et al., 2019; Guo et al., 2020; Huang et al., 2022; Macías- Rodríguez et al., 2020; Nosenko et al., 2023; Rajani et al., 2021; Ruangwong et al., 2021). It is already well-known that VOCs are pivotal in orchestrating inter-species and inter-kingdom signaling within the rhizosphere, a dynamic environment, where various organisms interact (Faure et al., 2009; Werner et al., 2016). They exert influence on growth, defense responses, and behavior of other organisms, with some VOCs exhibiting toxic properties (Hacquard & Schadt, 2015; Sivaprakasam Padmanaban et al., 2022) . However, the underlying mechanisms behind these effects and perception mechanisms ar e poorly understood (Hacquard, 2017; Werner et al., 2016). In line with our results, earlier in vitro co-cultivation scenarios of different Trichoderma strains, including T. harzianum WM24a1, with the ECM L. bicolor already revealed a stronger inhibitory effect of the ECM on the biocontrol strain than the other way around (Guo et al., 2019). The highest VOC emission rate was thereby detected when the two fungi were separated by several cm from each other, indicat ing VOCs to be an important tool for long - range inter-species communication and recognition. The upregulation of several metabolic KEGG pathways associated to “Biosynthesis of secondary metabolites”, “Steroid biosynthesis”, “alpha -Linolenic acid metabolism ”, “Terpenoid backbone biosynthesis”, and “Fatty acid metabolism”, which we now identified in L. bicolor in our setup, might reflect the ECM’s response to increased VOC emission rates of T. harzianum already at a distance. The introduced olfactometer “rac e-tube”-like system now allowed us to observe a strongly accentuated directional growth behavior of Trichoderma spp. compared to the conventional plate confrontation assays, demonstrating the positive or negative chemotropism in response to different fungal partners already at a distance and over time. In contrast to plate assays, which lack the capability for directional growth selection, the new system facilitates investigations into interactions spanning longer distances, enabling a choice of growth direction, as it would be the case under most natural conditions. The growth direction is influenced by soluble prey -derived molecules that are involved in fungal chemotropism by acting either as a chemoattractant (positive chemotropism) or as a chemorepellent (negative chemotropism) (Kullnig et al., 2000; Moreno -Ruiz et al., 2021) . Our experiments revealed consistent directional growth behavior of both Trichoderma strains towards the plant pathogens and away from ECM, even in the absence of soluble biochemicals or metabolites, was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 31 suggesting a significant role for VOCs in shaping the overall perception and observed physiological response. This underscores the importance of VOCs as potent mediators within the system, exerting a substantial impact on the inter -species interactions. Therefore, the developed olfactometer “race tube” -like system now enables further investigation of VOC- based fungal interactions and perceptions to screen for chemotropic growth. Transcriptomic differences in T. harzianum and L. bicolor The complex process of mycoparasitism is initiated by prey recognition, directed chemotrophic growth towards the prey, followed by direct attack through (bio -)chemical and physical mechanisms, ultimately leading to death and nutrient release (Chet et al., 1981; Harman, 2006; Moreno-Ruiz et al., 2020; Steindorff et al., 2014) . Importantly, the process is signal-dependent, relying on specific inter-species recognition mechanisms (Moreno-Ruiz et al., 2020; Sarm a et al., 2014; Zeilinger & Omann, 2007) . As the fungi are growing towards each other, they are constantly sensing their environment, and specific downstream signalling cascades are initiated based on the given abiotic and biotic conditions (Bahn et al., 2007; Hinterdobler et al., 2021; Turrà & Di Pietro, 2015; Zeilinger & Atanasova, 2020) . Transcriptomic analysis revealed plenty of common DEGs in T. harzianum during confrontation with the pathogens, while the confrontation with L. bicolor led to distinct - and much more moderate - expression patterns. These observations confirm the lifestyle-specific recognition between the fungi on the mo lecular level , leading to staging of a rapid and conserved mycoparasitic attack in case of the pathogens and literally a much more “relaxed” communication process in case of the ECM. Trichoderma spp. produce extracellular chitinases and proteases in a cons titutive manner, leading to enzymatically released chito - oligosaccharides and oligo-peptides in the presence of potential prey, which are sensed and initiate the expression of mycoparasitism-related genes (Benítez et al., 2005; Brunner et al., 2003; Druzhinina et al., 2011; las Mercedes Dana et al., 2001; Seidl et al., 2009) . During confrontation with A. alternata and F. graminearum three chitinases, belonging to the hydrolase family 18, were upregulated in T. harzianum already at distance (three days, MC), while the presence of L. bicolor did not activate these genes. Additionally, several gene s annotated with hydrolase activity, such as an endo -1,3-β-D-glucanase (M431DRAFT_479664) and a peptidase S1 domain -containing protein (M431DRAFT_526221), presumably involved in fungal cell wall degradation and proteolysis, were induced during confrontations with A. alternata and F. graminearum, but downregulated was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 32 during confrontation with L. bicolor . These results are in line with another comparative transcriptomic analysis that revealed a clear upregulation of mycoparasitism-related genes in T. atroviride, T. reesei, T. virens, as well as T. harzianum before direct physical contact with a fungal prey (Atanasova et al., 2013; Wang et al., 2024). The absence of induction of those genes during confrontation with L. bicolor is speaking in favour of a non -aggressive interaction. Nonetheless, the question whether this is the re sult of a self -governed decision by Trichoderma or is enforced by repressive or inhibitory metabolites produced by L. bicolor remains open so far. However, L. bicolor displayed only minimal transcriptomic changes in this situation, suggesting a lack of aggressive adaptations. Next to VOCs, signalling molecules and prey-derived oligo-peptides are assumed to act as ligands for G -protein-coupled receptors (GPCRs) that are involved in transduction of extracellular signals to intracellular-signaling networks in fungi (Xue et al., 2008) and part of the prey-sensing cascade in Trichoderma (Brunner et al., 2008; Hinterdobler et al., 2021; Lin et al., 2019; Seidl et al., 2009; Zeilinger & Atanasova, 2020) . Signal reception triggers downstream events via signal transduction mechanisms (Bahn et al., 2007; Carreras - Villaseñor et al., 2012) , which play a pivotal role in orchestrating the expression of specific sets of genes that govern the ultimate outcome of the interaction between two fungal species (Sarma et al., 2014) . During MC, we identified three upregulated genes (LACBIDRAFT_240638; LACBIDRAFT_246709; LACBIDRAFT_248257) in L. bicolor in presence of T. harzianum, which are annotated with FunCat categories “GPCR signalling”, “cellular communication”, and “signal transduction mechanism”. Furthermore, the upregulation of transcription factor Nrg1 in L. bicolor , has a putative function in carbon catabolite repression (Daguerre et al., 2017) and is involved in gene regulation of stress - response to salt and oxidative stress in Saccaromyces cerevisiae and Ustilago maydis (Sánchez-Arreguin et al., 2021). Also in T. harzianum , a rhodopsin domain -containing protein (M431DRAFT_155394) belonging to the GPCR rhodopsin family A (Harmar, 2001), was significantly downregulated in presence of L. bicolor and induced during confrontation with the pathogens. Additionally, a 3’,5’-cyclic-nucleotide phosphodiesterase (M431DRAFT_74093) was found to be significantly upregulated in T. harzianum during MC with L. bicolor, indicating modulation of intracellular levels of cyclic nucleotides, such as cyclic adenosine 3’,5’ monophosphate (cAMP). Those was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 33 messenger molecules are synthesized from ATP by adenylate cyclase activity and activate the cAMP -dependent protein kinase, leading to gene expression regulation by phosphorylation of e.g. transcription factors (Sun et al., 2022) . Biogenic VOCs emitted by post-harvested tomatoes, specifically ethylene and benzaldehyde, were identified as active compounds found to be tightly bound to GPCRs in B. cinerea, leading to a lack of signal transduction to the cAMP pathway, resulting in reduced pathogenicity of the plant -pathogen (Lin et al., 2019). The detected differential regulation of signal transduction -related genes in L. bicolor and T. harzianum, as well as the altered VOC emission profiles observed by Guo et al. (2019) during co-cultivation emphasize a VOC-mediated inter-species interaction and GPCRs signal transduction, with a distinct outcome of negative chemotropism and no induction of mycoparasitism-related cascades in T. harzianum. Signal-peptide containing proteins have already been described as effectors in beneficial plant-fungus interactions (Plett et al., 2011) , as well as in fungus -fungus interspecies interactions (Feldman et al., 2020) . Interestingly, we identified the un ique upregulation of a small-secreted protein (SSP) (M431DRAFT_96469) in T. harzianum in the presence of L. bicolor, while it was downregulated during confrontation with A. alternata and F. graminearum during MC . This SSP is a homologue (>90% sequence iden tity) of the cysteine-rich effector Tsp1 in T. virens, which was found to be induced in the presence of maize (Lamdan et al., 2015) and banana roots (Muthukathan et al., 2020) , indicating an involvement in Trichoderma-plant interaction and plant defence modulation. Gupta et al. (2021) analysed the function of Tsp1 in T. virens and identified structural similarity with the two fungal effector proteins PevD1 (Bu et al., 2014) and Alt a1 (Chruszcz et al., 2012), which are both interacting with plant defence proteins. The upregulation of this effector in T. harzianum confrontation with L. bicolor is a new finding and suggests an additional involvement in fungal inter-species interactions. Also in L. bicolo r, several short signal peptide -containing proteins were significantly upregulated during confrontation with T. harzianum, which might modulate the interaction by acting as secreted effector proteins. While ECM fungi are characterized by a restricted number of carbohydrate-active enzymes (CAZymes), their repertoires of secreted proteases and lipases is similar to saprotrophic fungi, and their secretomes are enriched in SSPs (Pellegrin et al., 2015) . Further studies are needed to investigate the role of those detected was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 17, 2024. ; https://doi.org/10.1101/2024.04.17.589723doi: bioRxiv preprint 34 and potentially secreted proteins in L. bicolor . Moreover, considering the plant roots as holobiont, further investigation into the influence of th e host plant on the interaction of T. harzianum and L. bicolor on the mycorrhized roots is required to gain further insights into the fungus-fungus signaling mechanisms and the influence of the plant on the overall outcome of the tripartite interaction. Concluding, we explored the complex interactions between Trichoderma spp. and fungal partners of different lifestyles, including two plant -beneficial ectomycorrhizal fungi (L. bicolor and H. cylindrosporum), and two plant -pathogenic fungi (F. graminearum and A. alternata), all potentially interacting within the rhizosphere of poplars. The present work allows insight into the distinct interactions of Trichoderma with ECM or pathogens , and sheds light on the multifaceted responses of Trichoderma towards root-associated fungi of different lifestyles , speaking in favor of a clear potential to distinguish between plant’s friend and foes during mycoparasitic confrontations. The described phenomenon of ECM avoidance already at a distance highlights the potential of Trichoderma spp. as a promising biocontrol agent and plant biofertilizer, while emphasizing the complexities of its interactions with various fungal associates. Acknowledgments The authors sincerely thank Karin Pritsch (H elmholtz Munich, Research Unit Environmental Simulation) for valuable advice and Sascha Schäuble (Department of Microbiome Dynamics, Hans-Knöll-Institute) for support with gene ontology analysis. Furthermore, we thank Franziska Vorwerk for excellent technical assistance. Competing interests The authors declare that they have no competing interests. Author contributions JPB, MR, PS and TK planned and designed the research. PS performed the experiments. PS, PBSP and JK analyzed data. PS wrote the manuscript. JPB, TK, MR and JPS reviewed and edited the manuscript. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 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