Exploring metabolic interaction between Ophiostoma novo-ulmi and Geosmithia spp

Exploring metabolic interaction between Ophiostoma novo-ulmi and Geosmithia spp | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring metabolic interaction between Ophiostoma novo-ulmi and Geosmithia spp Alessia Lucia Pepori, Hari Berto, Alessandra Gionni, Nicola Luchi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8842177/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Dutch elm disease (DED), caused by the invasive vascular pathogen Ophiostoma novo-ulmi , one of the most devastating pandemics affecting elms. Within beetle galleries and on elm bark beetle vectors, O. novo-ulmi co-occurs with fungi of the genus Geosmithia , yet the functional significance of this association remains poorly understood. This study investigates metabolic interactions between O. novo-ulmi and Geosmithia spp. using in vitro dual-culture experiments and phenotype microarray analysis to elucidate ecological mechanisms potentially influencing disease development and vector ecology. Dual-culture assays on dH 2 O agar revealed that O. novo-ulmi exhibited significantly enhanced radial growth rates when cultured in proximity to Geosmithia isolates. Phenotypic microarray analysis revealed distinct metabolic strategies: O. novo-ulmi utilised substrates within 12–24 hours and preferred polyols and simple carbohydrates, while G. pumila adopted a progressive colonisation strategy, ultimately utilising 92 substrates in 90 hours and demonstrating greater utilisation of nitrogen-enriched substrates, nucleosides and complex organic acids. Fungal competitiveness analysis indicated G. pumila superiority, yet O. novo-ulmi showed greater growth facilitation in co-culture, suggesting asymmetric mutualism through non-nutritional mechanisms. These results also suggest that physical proximity between O. novo-ulmi and Geosmithia spp. establishes a localized metabolic interface enabling chemical signaling and nutrient exchange. This mutualistic interaction, demonstrated by the increased growth of O. novo-ulmi in co-culture without suppressing Geosmithia spp., likely confers selective advantages that explain the evolutionary success of their ecological partnership. Within naturally colonised host tissues, such metabolic interaction may enhance pathogen establishment and persistence, with implications for disease epidemiology, beetle vector ecology, and potential disease management strategies. Ophiostoma novo-ulmi Geosmithia spp. metabolic interactions Dutch elm disease Biolog FF Plates carbon substrate utilization dual growth rate phenotyping Figures Figure 1 Figure 2 Figure 3 Introduction Since the early 20th century, European elms ( Ulmus spp.) have been severely threatened by Dutch elm disease (DED), a lethal vascular wilt caused by some invasive fungal pathogens of putative Asian origin belonging to the genus Ophiostoma , namely O. ulmi and O. novo-ulmi . The first epidemic wave was triggered by O. ulmi , that was later replaced by a more aggressive species, O. novo-ulmi , with two subspecies: O. novo-ulmi subsp. novo-ulmi and subsp. americana , which caused a second and more deadly pandemic. The rapid spread of DED is driven by a complex ecological network involving host species, fungal pathogens, bark beetles as vectors (primarily of the genus Scolytus) , phoretic mites, virus-like elements such as the d-factor, and other associated microorganisms. Recent research has proven the constant presence of fungi belonging to the genus Geosmithia ( Ascomycota : Hypocreales , Bionectriaceae ) within the insect galleries and on insect bodies, where they occupy the same ecosystem, and use similar dispersal mechanisms to those of O. novo-ulmi during most of their life cycle [ 1 – 5 ]. Extensive horizontal gene transfer (HGT) of the cerato-ulmin (cu) gene between O. novo-ulmi and several Geosmithia species has also been found between these two fungi [ 6 , 7 ]. This genetic exchange reveals a substantially more intricate interaction than simple coexistence; consequently, early studies [ 1 ] investigating the nature of this association within the DED pathosystem have proposed a potential hyperparasitic relationship between Geosmithia spp. and O. novo-ulmi . Further research [ 3 ] has further substantiated the frequent co-occurrence of Geosmithia spp. and O. novo-ulmi throughout the DED cycle, while revealing marked variation in their relative abundance according to the epidemiological stage of the disease in natural forest stands. Although Geosmithia does not exhibit any characteristics that suggest a close association with insects, Geosmithia species are closely related to beetles [ 8 ]. The ecological distribution of Geosmithia species reflects a spectrum of host associations and colonization strategies. While many species function as ecological generalists on hardwood trees, associating opportunistically with multiple bark beetle vectors, some have evolved specialized relationships with specific hosts and insect vectors. For instance, G. morbida has become functionally dependent on the walnut twig beetle ( Pityophthorus juglandis ) for dispersal and has evolved as a pathogen of black walnut ( Juglans nigra ), causing Thousand Cankers Disease, now spreading across the USA and Europe [ 9 , 10 ]. Some species have evolved as nutritional ambrosia fungi, establishing obligate mutualistic symbioses with their beetle vectors and colonizing beetle galleries as the primary food source for developing larvae [ 11 ]. This functional diversity, ranging from commensal habitat-sharers to specialized nutritional symbionts to pathogens, underscores the ecological complexity of Geosmithia -beetle-host plant interactions and supports the hypothesis that the functional role of Geosmithia species also within the DED pathosystem extends beyond simple opportunistic co-colonization. Based on these findings, we here investigated the metabolic activity and interactions of the two fungal species in vitro co-culture systems, with the aim of better understanding the mechanisms underlying the complex tri-trophic interactions involving the host ( Ulmus spp.), the fungal component ( Ophiostoma novo-ulmi and Geosmithia spp.), and its vectors (elm bark beetles). Microbial co-cultures better simulate the complex communities found in nature than monocultures, as microorganisms live in complex microbial communities in the natural environment. Microorganisms, plants, and the environment interact, share, and exchange metabolic processes and signals, and play a role of synergy or competitive antagonism [ 12 , 13 ]. The Phenotype Microarray ( ) system (Biolog Inc., Hayward, CA, USA) provides a practical, user-friendly, and cost-effective method for rapidly assessing fungal niche overlap and catabolic versatility by evaluating growth across a wide range of carbon sources [ 14 – 17 ]. Originally developed for bacteria, the system has been successfully adapted for fungi, including endophytes [ 18 , 19 ], to optimize growth conditions and secondary metabolite production [ 20 – 22 ]. In this paper, we adopt this approach to explore how metabolic-level fungal interactions may influence their ecological roles within the DED pathosystem and how these interactions could impact disease development and potential management strategies. In addition, an in vitro dual fungal growth trial onto a minimal substrate was also used to assess the effect of the interactions between the two fungal species in the exploitation of resources. The PM results will also be used to test the ability of the two fungi to compete for carbon resources, and the data obtained from the carbon utilization tests will allow the creation of a niche overlap index (NOI) [ 22 ] and also the ability to compete with each other [ 20 ]. Materials and Methods 1.1 Fungal Growth Rate in Dual Culture Eight Ophiostoma novo-ulmi isolates, four of which were O. novo-ulmi americana (NAN) and four O. novo-ulmi novo-ulmi (EAN), and twelve Geosmithia spp. isolates (selected among the main species of Geosmithia present on elm trees) were grown in dual culture (Table S1 ). For each fungal combination, three Petri dishes (90-mm diameter) filled with 25-ml dH 2 O agar substrate were inoculated by placing two 6-mm diameter mycelial plugs (one of Geosmithia spp. and one of Ophiostoma novo-ulmi ), obtained from the edges of actively growing fungal cultures, about 1 cm apart from each other near to the center of dish. Cultures were incubated in the dark at 24°C, and two radii of each colony on the growing edge opposite to the other fungus were measured after 48 h, 3, 5, and 8 days. Three plates per isolate were inoculated with two identical plugs as a control. Daily radial growth rates were compared by one-way ANOVA (Statistica 10, StatSoft Inc.). 1.2 Fungal metabolic potential analyses – FF Plate™ The phenotypic analysis was achieved using OmniLog® ID System multiplate reader (Biolog Inc., Hayward, CA, United States). Phenotype microarray experiments were performed using FF Biolog MicroArray™ plates to cover a broad spectrum of carbon substrates. Every microarray plate contains 95 low-molecular-weight carbon sources from biochemical groups such as: amines/amides (n = 6), amino acids (n = 13), carbohydrates (n = 44), carboxylic acids (n = 17), polymers (n = 5) and miscellaneous/other (n = 10) [ 17 ]. Each plate also contains water as a negative control (n = 1). FF plates were stored at 4°C until needed. Geosmithia pumila (IVV7) and O. novo-ulmi (ONU EAN H328), were selected for this trial since they are the most representative isolates of the species within the DED pathosystem, have been studied most extensively in recent years [ 1 , 2 , 6 , 7 , 24 , 25 , 28 – 31 ]. Three different phenotypic analyses were performed by inoculating the plates by i) O. novo-ulmi ; ii) G. pumila ; and their co-inoculum iii) O. novo-ulmi + G. pumila. Before inoculation, fungi were cultivated on MEA for at least one week at 25°C in the dark to obtain an adequate amount of fungal material from the mycelial growth phase. The superficial layer of the mycelial mat was scraped with a sterile cotton swab to transfer fungal biomass from the colonies (a mixture of spores and mycelial cells) into sterile glass tubes containing 12 ml of an inoculating fluid provided by Biolog. A tissue grinder (Kontes® Duall® 21, Kimble Chase) was used to gently disrupt the fungal biomass to make the inoculum more homogenous. The same suspensions were used to prepare both the single and the co-inoculum. The transmittance of each inoculum was adjusted to 75% by adding more fungal biomass or inoculation fluid as required. The co-inoculum consisted in a mixture of equal volumes of the single strains spores’ suspensions (1:1 ratio), which resulted, as well, in a final optical transmission of 75%. Three replica plates were prepared for each of the three analyses: O. novo-ulmi , O. novo-ulmi and G. pumila (double culture), and G. pumila (monoculture). The inoculated plates were kept at 25°C in darkness in the OmniLog® ID System multiplate reader (Biolog Inc., Hayward, CA, United States). Fungal growth was spectrophotometrically measured as optical density (OD) at 595 nm for 5 days, every 15 min, by the built-in camera and saved as OmniLog units generated by the Biolog® OmniLog PM software. The OD values of each isolate on the different substrates were normalized against those in the control well [ 16 ]. Data analysis for fungal strains’ metabolic profiling was conducted using Kinetic and Parametric software (Biolog). Phenotypes were determined based on the area under the kinetic curve of dye formation [ 14 ]. Heatmaps dendrogram was performed to show the correlation between the three treatments in relation to the utilization of C-sources from the FF microplates analyses. Furthermore, a comprehensive assessment of synergistic substrate utilization was conducted by measuring the difference between substrate consumption in co-cultures of Onu and G. pumila compared to their respective monocultures. Synergy was calculated as: synergy = ((ONU + G. pumila ) − (ONU monoculture + G. pumila monoculture))/2, as used by [ 32 ], where positive values indicate synergistic utilization (substrate consumption in co-culture exceeds the sum of individual monocultures) and negative values indicate negative interactions. Statistical analysis Daily fungal radial growth rates were compared by one-way ANOVA (Statistica 10, StatSoft Inc.) with Duncan test (p < 0.05). For the phenotypic data, an ANOVA was conducted to assess statistical differences among the groups. When the ANOVA indicated a significant effect (p < 0.05), Tukey’s Honest Significant Difference (HSD) post‑hoc test was applied to identify pairwise differences among the three inoculums. The statistical analyses were performed on raw data of absorbance values at 90 h. Then, the raw data were normalized to 0–1 range to perform a principal component analysis (PCA). The was employed to assess the metabolic differentiation between the three inoculums. All analysis were performed using R version 4.4.2 (R Core Team, 2024). Ecological analyses The niche overlap indices (NOI) were calculated according to Lee and Magan [ 22 ] and Wilson and Lindow [ 33 ]. A NOI value of 0.9 or above indicates a high degree of niche overlap and a competitive disadvantage for the target fungus [ 22 ]. We also calculated the O. novo-ulmi and Geosmithia’ s ability to compete with each other (fungal competitiveness, FC) based on the formula by Blumenstein et al. [ 20 ]. If the value of this function is greater than 1.0, one fungus exhibits competitive superiority relative to the other. Results Fungal Growth Rate in Dual Culture on agar/water The growth rate of O. novo-ulmi strains was generally higher in dual culture with Geosmithia spp. strains compared to monoculture controls (Table 1 ). Across all dual culture tests, Ophiostoma strains showed significantly higher growth rates when grown in dual culture with each Geosmithia isolate tested, (Duncan test, p ≤ 0.05), despite growth occurred on nutritionally inert substrate (water agar). In particular, Onu EAN (H328) grows significantly more with all Geosmithia isolates tested (significant Duncan test, p ≤ 0.05). No significant differences in growth rates were observed between Geosmithia species when grown with Ophiostoma , apart from a few isolate-specific interactions, as shown in the table. Within each species, all strains grew at the same rate (non-significant Duncan test, p > 0.05). Table 1 Fungal growth rates in dual culture assays. Grey background indicates O. novo-ulmi growth in the presence of Geosmithia spp., while white background indicates Geosmithia growth in the presence of O. novo-ulmi strains. (+) indicates significantly increased growth compared to controls (Duncan's test, p ≤ 0.05); (−) indicates significantly decreased growth compared to controls (Duncan's test, p ≤ 0.05). Fungal growth rate G. pumila G. funiculosa G. langdonii G. omnicola G. ulmacea G. flava G. putterillii CNR39 IVV7 CNR102 CNR48 CNR31 CNR26 CNR105 CNR8 CNR32 CNR23 CNR120 CCF3342 Onu NAN H172 + + + + + + + 182E + + + + + OPH123 + + + + + + + OPH32A + + + + - + + + + Onu EAN CTK11 + + + + + + + - + R64 + + + + + + + + + + H327 + + + + + + + H328 + + + + + + + + + + + + Biolog Results Analysis The three phenotypic analyses carbon substrate utilization were compared revealing distinct metabolic patterns among the 95 substrates. Substrate activation was analyzed by quantifying the number of substrates with metabolic activity (OD > 10 units) at each timepoint up to 90 hours. Three distinct temporal phases characterized the metabolic dynamics of the two fungal species and their co-culture. During the early phase (12–24 hours), G. pumila initiated substrate utilization slowly, with only 13 substrates at 12 hours, progressively increasing to 50 by 24 hours. O. novo-ulmi demonstrated a more rapid metabolic onset, utilizing 46 substrates at 12 hours and reaching 70 by 24 hours. The co-culture of O. novo-ulmi & G. pumila displayed intermediate metabolic activity, with 18 substrates at 12 hours and 63 substrates by 24 hours (Fig. 1 a). During the intermediate phase (36–48 hours), G. pumila accelerated its expansion, while O. novo-ulmi stabilised a plateau. The co-culture matched G. pumila performance. During the late phase (60–90 hours), G. pumila and the co-colture continued its expansion (up to 92 and 88 substrates, respectively), while O. novo-ulmi remained stable (77 substrates). Analysis of hourly substrate consumption rates (OD mean/hours) revealed distinct metabolic dynamics among the three treatments. O. novo-ulmi exhibited a pronounced peak of approximately 4.2 at 24 hours then decreased rapidly after, indicating intense but transient metabolic activity. In contrast, G. pumila and the combined culture maintained relatively stable and lower consumption rates (2.0–2.5) throughout the observation period. After 24–36 hours, all treatments showed a gradual decline in consumption rates, converging toward 1.5–1.8 by 90 hours, suggesting a shift from active substrate utilization to a stationary metabolic phase (Fig. 1 b). Regarding average carbon substrate utilization, G. pumila demonstrated the highest metabolic capacity, achieving approximately 155 OD units by 90 hours despite a slower initial growth phase. Conversely, O. novo-ulmi exhibited rapid substrate utilization during the first 48 hours but subsequently plateaued at lower levels than the other treatments (Fig. 1 c). Analysis of individual substrate categories revealed that the G. pumila strain demonstrated exclusive or superior utilization of complex nitrogen substrates (Fig. 2 ). N-Acetyl-L-Glutamic Acid, L-Pyroglutamic Acid, N-Acetyl-D-Galactosamine and Sebacic Acid were completely unused by O. novo-ulmi but fully utilized in G. pumila treatments and robustly exploited in co-culture (OD 90–165). The use of L-phenylalanine showed a marked rise in co-culture, indicating an increase in the catabolism of aromatic amino acids with greater metabolic diversity. In contrast, the utilization of N-acetyl-D-mannosamine and Lactulose was exploited by both monocultures but not by the co-culture, suggesting direct metabolic antagonism or enzymatic competition. Nucleosides and Nucleotides. Adenosine showed the most striking synergistic response, remaining nearly unused by ONU in monoculture (OD ~ 10–25) yet activating to high levels in co-culture (OD 120–195 by 48 h), demonstrating direct metabolic facilitation of the co-culture. Similarly, Uridine showed increased utilization level in co-culture, indicating a G. pumila -mediated improvement. Organic Acids. Several carboxylic acids were exclusively or preferentially used by G. pumila . Bromosuccinic Acid, p-Hydroxyphenylacetic Acid, and D-Saccharic Acid remained unexploited by O. novo-ulmi alone but became metabolically available in dual culture. Polyols and Sugar Alcohols . O. novo-ulmi showed preferential utilization of polyols in monoculture, which were antagonistically suppressed in dual culture. D-Mannitol, L-Sorbose, Xylitol, Adonitol and Maltitol all exhibited marked suppression in co-culture (OD ~ 50–100 vs. 150–200 in O. novo-ulmi monoculture), suggesting competitive exclusion by G. pumila . G. pumila enhanced the utilization of atypical monosaccharides. D-Ribose, D-Tagatose, D-Gluconic Acid, and Stachyose were scarcely used by O. novo-ulmi , while they reached high levels of utilisation in dual culture and G. pumila monoculture. Conversely, D-Arabinose and L-Fucose remained poorly utilized across all treatments, suggesting limited enzymatic capacity in both species. Cyclodextrin metabolism was G. pumila -exclusive: α-Cyclodextrin and β-Cyclodextrin were metabolized just by G. pumila . Sedoheptulosan utilization was absent in O. novo-ulmi (OD = 0) but dramatically increased in co-culture with G. pumila (OD = 95.58 vs. 10.39 G. pumila monoculture), indicating synergistic metabolic activation. Comprehensive analysis of 95 distinct chemical substrates revealed substantial heterogeneity in synergistic responses (Table S2). The most pronounced positive synergistic responses were observed for carbohydrate-derived compounds and sugar alcohols. Sedoheptulosan exhibited the strongest synergy (+ 90.39), representing a 90-unit increase in net substrate utilization in co-culture. Other highly synergistic substrates included α-methyl-D-glucoside (+ 84.50), N-acetyl-L-glutamic acid (+ 84.30), and L-pyroglutamic acid (+ 80.88). Conversely, several substrates exhibited strong negative synergy, indicating competitive inhibition or reduced substrate accessibility under co-culture conditions. Xylitol displayed the strongest antagonistic response (− 82.41), followed by D-glucosamine (− 45.62) and maltitol (− 44.63). Additional substantially antagonistic substrates included D-psicose (− 40.63), succinic acid (− 35.83), and salicin (− 33.26). All those difference are statistically significant (p < 0.05). PCA Biplot Analysis of Metabolic Differentiation Principal component analysis of substrate utilisation patterns revealed distinct metabolic separation among O. novo-ulmi , G. pumila , and their co-culture ( O. novo-ulmi & G. pumila ), with the first two dimensions explaining 99.6% of total variance (PC1: 67.4%, PC2: 32.2%) (Fig. 3 ). The biplot demonstrated clear spatial clustering of the three treatments, reflecting fundamental differences in substrate preference and enzymatic capacity. Onu occupied the upper-right quadrant, characterised by strong associations with polyols (D-mannitol, xylitol, adonitol, maltitol) and simple carbohydrates. In contrast, G. pumila positioned in the upper-left quadrant, showing preferential utilisation of nitrogen-enriched substrates (L-pyroglutamic acid, N-acetyl-L-glutamic acid, N-acetyl-D-galactosamine), complex organic acids (bromosuccinic acid, p-hydroxyphenylacetic acid), nucleosides (adenosine, uridine), and exclusive utilisation of cyclodextrins (α and β), and were completely unexploited by Onu. The co-culture occupied an intermediate central position, reflecting a metabolically integrated system rather than dominance by either partner. Compounds contributing most significantly to PC1 included N-acetyl D-glucosamine, salicin, and L-ornithine, indicating carbohydrate and amino acid metabolism as primary discriminatory factors. Compounds clustering along PC2 included various hexoses, pentoses, and organic acids, distinguishing strains based on specific metabolic pathways for sugar utilization and organic acid production. Ecological analyses The analysis of the nutritional profile of the three fungal strains revealed significant differences in their substrate utilization capacity. Niche overlap index (NOI) analysis revealed moderate nutritional overlap among the strains: O. novo-ulmi shared 74% of its substrates with G. pumila , while G. pumila exhibited 82% overlap with Ophiostoma 's substrates. These results suggest that the two fungi occupy partially overlapping but distinct nutritional niches. Regarding fungal competitiveness (FC) according to the Blumenstein et al. [ 20 ] model, O. novo-ulmi demonstrated an FC of 0.89 against G. pumila , indicating moderate competitive capacity for nutritional resources. In contrast, G. pumila showed an FC of 1.15 against O. novo-ulm i, suggesting superior competitiveness in the context of nutritional competition. These findings indicate that, although there is substantial metabolic overlap among the strains, G. pumila possesses a greater capacity to compete for shared nutritional substrates. Discussion The increased growth of O. novo-ulmi in dual culture with Geosmithia spp. on nutritionally inert water agar substrate presents an unexpected result; despite the competitive superiority of Geosmithia (FC = 1.15 versus 0.89), O. novo-ulmi shows greater growth when physically close to Geosmithia than in monoculture controls. This observation is consistent with its ecological specialization as a wood-colonizing pathogen that depends on complex organic substrates [ 34 ]. The absence of significant reciprocal growth enhancement for Geosmithia species in co-culture, combined with the intermediate FC values, suggests an asymmetric mutualism in which O. novo-ulmi benefits from physical proximity and possibly biochemical facilitation from the presence of the other fungus. The data indicate that physical contact between the two fungi establishes a localized metabolic interface through which selective nutrient exchange and chemical signaling occur [ 35 ]. Although phenotypic analysis shows that G. pumila utilises more substrates than O. novo-ulmi (93.68% versus 83.16% within 90 hours), the growth of O. novo-ulmi in dual culture on nutrient-free substrate indicates that facilitation operates through non-nutritional mechanisms rather than nutrient supply. This is consistent with the current understanding that microbial coexistence often depends on chemical interactions and metabolic plasticity rather than simple competition for resources [ 36 ]. The growth enhancement observed on nutrient-depleted substrate suggests that Geosmithia spp. may facilitate O. novo-ulmi through non-nutritional mechanisms, including modulation of the local microenvironment, rather than through provision of exogenous nutrients [ 37 ]. The phenotypic analysis provides mechanistic insight into how this metabolic interaction operates. Although our study was focused to two fungal isolates, these strains were selected based on their extensive characterisation in previous studies, including documented events of HGT [ 1 , 2 , 6 , 7 , 24 , 25 , 28 – 31 ]. The two fungal species exhibited distinct metabolic strategies. O. novo-ulmi demonstrated rapid utilisation of a greater number of substrates in the early phase (12–24 hrs), while G. pumila showed colonisation on fewer substrates, followed by accelerated expansion until reaching the highest number of activated substrates, at the late stage (60–90 hrs). This difference in metabolic strategy can be explained by the nature of the fungal pathogens. O. novo-ulmi employs a rapid-exploitation strategy on preferred substrates, maximising fitness within a narrow time window, presumably to maintain a high level of pathogenicity towards the host plant, a pattern also observed in soil pathogens such as Pythium aphanidermatum and Fusarium oxysporum f.sp. radicis-lycopersic i [ 38 ]. In contrast, G. pumila appears to adopt a more conservative metabolic strategy characterised by a progressive capacity to utilise the substrate throughout the observation period. Co-culture displays an intermediate metabolic profile that progressively converges toward the G. pumila phenotype, exemplifying the well-established principle that metabolically superior competitors eclipse early colonizers in microbial communities [ 39 ]. The metabolic synergy, as utilisation of adenosine, is a widespread phenomenon in microbial communities [ 40 ]. O. novo-ulmi shows minimal adenosine metabolism in monoculture (OD ~ 10–25 at 48 hours) but achieves robust utilisation in co-culture (OD 120–195 at 48 hours), with a 5–19-fold increase. We observe a similar trend for adenosine 5'-monophosphate substrate. We hypothesize that this facilitation does not result from competitive inhibition, as G. pumila effectively metabolises adenosines independently. Rather, the data indicate that G. pumila could enzymatically degrade adenosine into other degradation products accessible to the metabolism of O. novo-ulmi , as reported for other fungi [ 41 , 42 ]. O. novo-ulmi showed a preferential utilisation of polyols (D-mannitol, L-sorbose, xylitol, adonitol, maltitol) in monoculture, with OD values ranging from 150 to 200. In dual culture, the utilisation of these substrates was significantly suppressed (OD ~ 50–100), suggesting competitive exclusion by G. pumila . Similarly, N-acetyl-D-mannosamine was preferentially utilised by O. novo-ulmi alone but antagonistically suppressed in co-culture. The increased growth of O. novo-ulmi on nutrient-depleted agar despite the suppression of polyols indicates that growth facilitation does not depend on the utilisation of these compounds. Instead, O. novo-ulmi appears to shift its metabolic investment towards alternative responses, such as the activation of stress pathways or the mobilisation of internal reserves, independent of external nutrients. This metabolic flexibility is consistent with documented stress responses in pathogenic fungi [ 43 ]. These findings reveal an interaction in which G. pumila competitively monopolises specific nutritional niches (polyols, certain amino sugars) while simultaneously facilitating O. novo-ulmi 's access to alternative and metabolically distinct substrates. Analysis of the niche overlap index (NOI) quantified this division: O. novo-ulmi shared 75.0% of its substrates with G. pumila , while the latter showed a 94% overlap with O. novo-ulmi substrates, an asymmetry that reflected Geosmithia 's broader metabolic capacity. These results, combined with some observed metabolic separation, suggest that the Ulmus - O. novo-ulmi - Geosmithia system is consistent with niche construction theory, according to which symbiotic partners construct niches through their metabolism and activities, creating a division that reflects both competitive and facilitating dynamics.[ 44 ]. Principal component analysis confirmed this niche differentiation, demonstrating a clear metabolic separation (PC1: 67.4%, PC2: 32.2%) in which O. novo-ulmi clustered with metabolic nodes associated with polyols, while Geosmithia associated with nitrogen-enriched substrates and nucleosides. The co-culture occupied an intermediate position, tending towards the G. pumila phenotype. Fungal competitiveness (FC) analysis revealed that Geosmithia (FC = 1.147) has a higher competitive capacity for shared nutritional resources than O. novo-ulmi (FC = 0.636), in line with field observations where Geosmithia often dominates in ecologically competitive contexts [ 1 , 3 , 7 ]. However, this nutritional competitiveness becomes functionally irrelevant on nutrient-poor substrates such as water/agar, where exogenous carbon and nitrogen are absent. This context dependence suggests that on nutrient-free substrate, Geosmithia likely facilitates O. novo-ulmi growth through non-nutritional mechanisms. Microbial interactions are regulated by multiple factors, including chemical, biological, physical, and genetic elements, with substrate and nutrient composition playing a key role in determining interaction patterns [ 45 ]. Furthermore, fungal pathogens raise local pH through ammonia production, improving stress tolerance and growth conditions [ 46 ]. Moreover, fungi subjected to osmotic stress accumulate protective compounds (glycerol, trehalose, proline) that help cells survive drying stress [ 47 , 48 ]. The co-presence of Geosmithia spp. may enhance the ability of O. novo-ulmi to produce these protective compounds, allowing for more efficient use of internal resources for growth. This is consistent with studies showing that in nutrient-poor environments, fungal coexistence is determined by non-nutritional factors such as pH modification and stress tolerance rather than direct competition [ 49 , 50 ]. The distinction between metabolic potential (as revealed by PM analysis with defined substrates) and in vitro growth on “poor” substrate is ecologically fundamental to understanding the Onu- Geosmithia spp. interaction within the DED pathosystem. In naturally colonised host tissues, microhabitat heterogeneity creates zones with variable nutrient availability: fresh xylem tissue provides abundant simple carbohydrates [ 51 ] preferred by Onu, while beetle excrement and degraded tissue offer nitrogen-enriched resources [ 52 ] exploited by Geosmithia spp.. It has been observed [ 53 ] that the growth of many species of Geosmithia is favoured by the urea, uric acid, and in part, ammonia, which demonstrates their ability to recycle nitrogen, probably originating from beetle excrement. Since the concentration of nitrogen in beetle galleries greatly affects their vitality [ 54 ], the presence of Geosmithia spp. for nitrogen recycling seems to be of great importance within the DED pathosystem. Pathogenic fungi, such as O. novo-ulmi , rapidly regulate their metabolism in response to available resources [ 43 ]. This metabolic flexibility allows Onu to increase overall growth despite limited access to its preferred substrates. Results indicate that Geosmithia species releases nitrogen-containing compounds and nucleosides into the co-culture, redirecting Onu's metabolism towards purine and pyrimidine metabolism, as reported by Chitty et al. [ 55 ] for human fungal pathogens. This metabolic shift is documented in nutrient-depleted fungal cells as a strategy to sustain essential biosynthetic processes and energy production [ 56 ]. The results of this research provide novel insights about the O. novo-ulmi - Geosmithia interaction, changing the perspective on the mycoparasitic interpretation of the interaction, in which morphological penetration of the hyphae was understood exclusively as predatory antagonism [ 1 ]. This study suggests that physical contact may establish a finely regulated metabolic exchange and chemical signalling, while the evolutionary stability of horizontal gene transfer events between these fungi ( cerato-ulmin gene transfer; [ 7 ]) supports long-term functional integration between these fungal species. This biological integration, combined with our metabolic data, supports the hypothesis that the two fungi have converged towards a form of functional symbiosis within the DED pathosystem. The distinction between metabolic potential (revealed by phenotypic analysis with microarrays with defined substrates) and growth on nutrient-poor substrates is fundamental from an ecological point of view: in naturally colonised host tissues, microhabitat heterogeneity creates zones with variable nutrient availability, and the ability of fungi to dynamically partition niches, exploit resources at different times, and facilitate growth in the absence of nutrients likely significantly contributes to their ecological success. The metabolic advantages deriving from their interaction are manifold: each fungus facilitates the other's access to otherwise inaccessible nutrient sources, including through a possible reciprocal exchange of metabolites and enzymatic activities, and O. novo-ulmi grows significantly more than in monoculture without affecting Geosmithia spp. growth. Collectively, these results suggest that their fungal coexistence may represent a mutualistic strategy conferring selective advantages over solitary survival, thus explaining the evolution of their ecological and biological interaction. Future investigations will be useful to characterise secondary metabolite profiles, pH dynamics, and osmolite production during co-culture growth to identify specific non-nutritional mechanisms driving the growth facilitation of O. novo-ulmi under nutrient-deficient conditions. Such investigations will deepen our understanding of fungal pathogenesis in the context of complex microbial communities and inform strategies for disease management in natural ecosystems. Declarations Ethics Approval No ethical approval was required for this study as it did not involve the use of any vertebrate animals or endangered insects. Competing interests The authors declare no competing interests. Funding We acknowledge financial support under the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, project: “Ulmus glabra protection in Italian peninsula – MONTANA”, CUP: B53D23012340006, published on 2.2.2022 by the Italian Ministry of University and Research (MUR), and funded by the European Union – NextGenerationEU. Author Contribution A.L.P. (Conceptualization, Data curation, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing, Funding acquisition, Project administration), H.B. (Data curation, Formal analysis, Investigation, Writing – review & editing), N.L. (Conceptualization, Writing – review & editing), A.G. (Investigation, Methodology), F.P. (Methodology, Writing – review & editing), A.S. (Conceptualization, Writing – original draft, Writing – review & editing). Data Availability Data will be made available on request. 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Curr Opin Insect Sci 39:27–34. https://doi.org/10.1016/j.cois.2019.12.005 Sarsan S, Pandiyan A, Rodhe AV, Jagavati S (2021) Synergistic Interactions Among Microbial Communities. In: Singh RP, Manchanda G, Bhattacharjee K, Panosyan H (eds) Microbes in Microbial Communities: Ecological and Applied Perspectives. Springer Singapore, Singapore, pp 1–37 St Leger RJ, Nelson JO, Screen SE (1999) The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity. Microbiology 145:2691–2699 Pérez-Llano Y, Rodríguez-Pupo EC, Druzhinina IS et al (2020) Stress reshapes the physiological response of halophile fungi to salinity. Cells 9:525 Davis DJ, Burlak C, Nicholas P (2000) Osmotic pressure of fungal compatible osmolytes. Mycol Res 104:800–804 Glassman SI, Wang IJ, Bruns TD (2017) Environmental filtering by pH and soil nutrients drives community assembly in fungi at fine spatial scales. Mol Ecol 26:6960–6973. https://doi.org/10.1111/mec.14414 Velez P, Espinosa-Asuar L, Figueroa M et al (2018) Nutrient dependent cross-kingdom interactions: fungi and bacteria from an oligotrophic desert oasis. Front Microbiol 9:1755. https://doi.org/10.3389/fmicb.2018.01755 Lehenberger M, Benkert M, Biedermann PH (2021) Ethanol-enriched substrate facilitates ambrosia beetle fungi, but inhibits their pathogens and fungal symbionts of bark beetles. Front Microbiol 11:590111 Ceja-Navarro JA, Karaoz U, Bill M et al (2019) Gut anatomical properties and microbial functional assembly promote lignocellulose deconstruction and colony subsistence of a wood-feeding beetle. Nat Microbiol 4:864–875 Veselská T, Skelton J, Kostovčík M et al (2019) Adaptive traits of bark and ambrosia beetle-associated fungi. Fungal Ecol 41:165–176. https://doi.org/10.1016/j.funeco.2019.06.005 Ayres MP, Wilkens RT, Ruel JJ et al (2000) Nitrogen budgets of phloem-feeding bark beetles with and without symbiotic fungi. Ecology 81:2198–2210 Chitty JL, Fraser JA (2017) Purine acquisition and synthesis by human fungal pathogens. Microorganisms 5:33 Fleck CB, Schöbel F, Brock M (2011) Nutrient acquisition by pathogenic fungi: nutrient availability, pathway regulation, and differences in substrate utilization. Int J Med Microbiol 301:400–407 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8842177","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591163596,"identity":"72c13c22-57eb-4a4f-8197-ef0e27677ac6","order_by":0,"name":"Alessia Lucia Pepori","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIie3PsQrCMBCA4SuBdgm4VgT7CpelRRCfRSg4iTgKDgZc1Wdxdavc0CXqKjiIuDpYXDo42CpFuiSODvkpTTJ8JAdgs/1jXvnD6oTd95JoCauTwYdoDasfCYzXNOaMsnzcg8hbbq+T8SGIWolDdw3xyR20OMbQWexjofAkNqu+4WHEQwZYfMdh2JR46qMyzBJQ45HlOAM830qyNxMkDj5HKm7hJUnMRJAbFrOkvLMYxUJiLNbKkYnSkHY6v2b5c9qOvN32Ip+9ABWj+0RDqjgCr/aO/AGUQ32JzWaz2eq9ADebSzpsOlOxAAAAAElFTkSuQmCC","orcid":"","institution":"National Research Council","correspondingAuthor":true,"prefix":"","firstName":"Alessia","middleName":"Lucia","lastName":"Pepori","suffix":""},{"id":591163597,"identity":"2fee31c9-7d1d-4169-ba6e-076aed42ef5d","order_by":1,"name":"Hari Berto","email":"","orcid":"","institution":"National Research Council","correspondingAuthor":false,"prefix":"","firstName":"Hari","middleName":"","lastName":"Berto","suffix":""},{"id":591163598,"identity":"867029f5-8c91-49b4-8657-06b933874890","order_by":2,"name":"Alessandra Gionni","email":"","orcid":"","institution":"National Research Council","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Gionni","suffix":""},{"id":591163599,"identity":"af75bb9b-0cb7-465a-932f-becf4243f674","order_by":3,"name":"Nicola Luchi","email":"","orcid":"","institution":"National Research Council","correspondingAuthor":false,"prefix":"","firstName":"Nicola","middleName":"","lastName":"Luchi","suffix":""},{"id":591163600,"identity":"075cf05b-123f-4800-a771-ba4fced55251","order_by":4,"name":"Francesco Pecori","email":"","orcid":"","institution":"National Research Council","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Pecori","suffix":""},{"id":591163601,"identity":"a911e134-24d6-4d3d-b2ee-024d8f8411b8","order_by":5,"name":"Alberto Santini","email":"","orcid":"","institution":"National Research Council","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Santini","suffix":""}],"badges":[],"createdAt":"2026-02-10 14:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8842177/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8842177/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102962855,"identity":"1fc1c16f-2546-42cf-8551-82e596da78a9","added_by":"auto","created_at":"2026-02-19 04:11:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":160949,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic carbon substrate utilization profiles of \u003cem\u003eGeosmithia pumila\u003c/em\u003e, \u003cem\u003eOphiostoma novo-ulmi\u003c/em\u003e, and their co-culture over 90 hours. (a) Number of substrates with metabolic activity (b) Hourly substrate consumption rates (OD mean/hours) (c) Mean OD values indicating total substrate utilization capacity.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8842177/v1/c6b02fb964b663aa0c3af63a.png"},{"id":104397298,"identity":"3504c089-ed46-4439-989e-78e1d8d389b2","added_by":"auto","created_at":"2026-03-11 11:46:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":365737,"visible":true,"origin":"","legend":"\u003cp\u003eComparative heatmap of \u003cem\u003eG. pumila\u003c/em\u003e (monoculture), \u003cem\u003eO. novo-ulmi\u003c/em\u003e \u0026amp; \u003cem\u003eG. pumila\u003c/em\u003e (dual culture), and \u003cem\u003eO. novo-ulmi\u003c/em\u003e (monoculture) strains grown on Biolog FF MicroPlates™ after 90 hours based on the utilization of 95 different carbon sources. The green to red color gradient indicates poor to high growth on the respective substrates.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8842177/v1/0b0bd6a46a3fad4442c7635c.png"},{"id":102778409,"identity":"95bc9a50-c3fd-4bff-a966-f76507337a24","added_by":"auto","created_at":"2026-02-16 14:29:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":313655,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) of 95 carbon substrates utilization after 90h of \u003cem\u003eG. pumila\u003c/em\u003e (monoculture), \u003cem\u003eO. novo-ulmi\u003c/em\u003e (monoculture) and \u003cem\u003eO. novo-ulmi\u003c/em\u003e \u0026amp; \u003cem\u003eG. pumila\u003c/em\u003e (co-colture). Each point represents a metabolite, coloured according to its loading on the principal components.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8842177/v1/9fd4a8b087be30b297d9c0f5.png"},{"id":104409958,"identity":"7a136253-7515-4e69-939a-8063eeef79a9","added_by":"auto","created_at":"2026-03-11 12:48:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1449672,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8842177/v1/cbd47279-89f7-4a88-9063-68563f5bf677.pdf"},{"id":102778406,"identity":"27e0d62f-e279-4854-9b0d-75b2acd5ae4c","added_by":"auto","created_at":"2026-02-16 14:29:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":24004,"visible":true,"origin":"","legend":"","description":"","filename":"Supplmat.docx","url":"https://assets-eu.researchsquare.com/files/rs-8842177/v1/0e51c6c11794271724f266de.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring metabolic interaction between Ophiostoma novo-ulmi and Geosmithia spp","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince the early 20th century, European elms (\u003cem\u003eUlmus\u003c/em\u003e spp.) have been severely threatened by Dutch elm disease (DED), a lethal vascular wilt caused by some invasive fungal pathogens of putative Asian origin belonging to the genus \u003cem\u003eOphiostoma\u003c/em\u003e, namely \u003cem\u003eO. ulmi\u003c/em\u003e and \u003cem\u003eO. novo-ulmi\u003c/em\u003e. The first epidemic wave was triggered by \u003cem\u003eO. ulmi\u003c/em\u003e, that was later replaced by a more aggressive species, \u003cem\u003eO. novo-ulmi\u003c/em\u003e, with two subspecies: \u003cem\u003eO. novo-ulmi\u003c/em\u003e subsp. \u003cem\u003enovo-ulmi\u003c/em\u003e and subsp. \u003cem\u003eamericana\u003c/em\u003e, which caused a second and more deadly pandemic. The rapid spread of DED is driven by a complex ecological network involving host species, fungal pathogens, bark beetles as vectors (primarily of the genus \u003cem\u003eScolytus)\u003c/em\u003e, phoretic mites, virus-like elements such as the d-factor, and other associated microorganisms. Recent research has proven the constant presence of fungi belonging to the genus \u003cem\u003eGeosmithia\u003c/em\u003e (\u003cem\u003eAscomycota\u003c/em\u003e: \u003cem\u003eHypocreales\u003c/em\u003e, \u003cem\u003eBionectriaceae\u003c/em\u003e) within the insect galleries and on insect bodies, where they occupy the same ecosystem, and use similar dispersal mechanisms to those of \u003cem\u003eO. novo-ulmi\u003c/em\u003e during most of their life cycle [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Extensive horizontal gene transfer (HGT) of the cerato-ulmin (cu) gene between \u003cem\u003eO. novo-ulmi\u003c/em\u003e and several \u003cem\u003eGeosmithia\u003c/em\u003e species has also been found between these two fungi [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This genetic exchange reveals a substantially more intricate interaction than simple coexistence; consequently, early studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] investigating the nature of this association within the DED pathosystem have proposed a potential hyperparasitic relationship between \u003cem\u003eGeosmithia\u003c/em\u003e spp. and \u003cem\u003eO. novo-ulmi\u003c/em\u003e. Further research [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] has further substantiated the frequent co-occurrence of \u003cem\u003eGeosmithia\u003c/em\u003e spp. and \u003cem\u003eO. novo-ulmi\u003c/em\u003e throughout the DED cycle, while revealing marked variation in their relative abundance according to the epidemiological stage of the disease in natural forest stands. Although Geosmithia does not exhibit any characteristics that suggest a close association with insects, Geosmithia species are closely related to beetles [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe ecological distribution of \u003cem\u003eGeosmithia\u003c/em\u003e species reflects a spectrum of host associations and colonization strategies. While many species function as ecological generalists on hardwood trees, associating opportunistically with multiple bark beetle vectors, some have evolved specialized relationships with specific hosts and insect vectors. For instance, \u003cem\u003eG. morbida\u003c/em\u003e has become functionally dependent on the walnut twig beetle (\u003cem\u003ePityophthorus juglandis\u003c/em\u003e) for dispersal and has evolved as a pathogen of black walnut (\u003cem\u003eJuglans nigra\u003c/em\u003e), causing Thousand Cankers Disease, now spreading across the USA and Europe [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Some species have evolved as nutritional ambrosia fungi, establishing obligate mutualistic symbioses with their beetle vectors and colonizing beetle galleries as the primary food source for developing larvae [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This functional diversity, ranging from commensal habitat-sharers to specialized nutritional symbionts to pathogens, underscores the ecological complexity of \u003cem\u003eGeosmithia\u003c/em\u003e-beetle-host plant interactions and supports the hypothesis that the functional role of \u003cem\u003eGeosmithia\u003c/em\u003e species also within the DED pathosystem extends beyond simple opportunistic co-colonization.\u003c/p\u003e \u003cp\u003eBased on these findings, we here investigated the metabolic activity and interactions of the two fungal species in vitro co-culture systems, with the aim of better understanding the mechanisms underlying the complex tri-trophic interactions involving the host (\u003cem\u003eUlmus\u003c/em\u003e spp.), the fungal component (\u003cem\u003eOphiostoma novo-ulmi\u003c/em\u003e and \u003cem\u003eGeosmithia\u003c/em\u003e spp.), and its vectors (elm bark beetles). Microbial co-cultures better simulate the complex communities found in nature than monocultures, as microorganisms live in complex microbial communities in the natural environment. Microorganisms, plants, and the environment interact, share, and exchange metabolic processes and signals, and play a role of synergy or competitive antagonism [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Phenotype Microarray ( ) system (Biolog Inc., Hayward, CA, USA) provides a practical, user-friendly, and cost-effective method for rapidly assessing fungal niche overlap and catabolic versatility by evaluating growth across a wide range of carbon sources [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Originally developed for bacteria, the system has been successfully adapted for fungi, including endophytes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], to optimize growth conditions and secondary metabolite production [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this paper, we adopt this approach to explore how metabolic-level fungal interactions may influence their ecological roles within the DED pathosystem and how these interactions could impact disease development and potential management strategies. In addition, an in vitro dual fungal growth trial onto a minimal substrate was also used to assess the effect of the interactions between the two fungal species in the exploitation of resources. The PM results will also be used to test the ability of the two fungi to compete for carbon resources, and the data obtained from the carbon utilization tests will allow the creation of a niche overlap index (NOI) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and also the ability to compete with each other [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e1.1 Fungal Growth Rate in Dual Culture\u003c/p\u003e\n\u003cp\u003eEight \u003cem\u003eOphiostoma novo-ulmi\u003c/em\u003e isolates, four of which were \u003cem\u003eO. novo-ulmi americana\u003c/em\u003e (NAN) and four \u003cem\u003eO. novo-ulmi novo-ulmi\u003c/em\u003e (EAN), and twelve \u003cem\u003eGeosmithia\u003c/em\u003e spp. isolates (selected among the main species of \u003cem\u003eGeosmithia\u003c/em\u003e present on elm trees) were grown in dual culture (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). For each fungal combination, three Petri dishes (90-mm diameter) filled with 25-ml dH\u003csub\u003e2\u003c/sub\u003eO agar substrate were inoculated by placing two 6-mm diameter mycelial plugs (one of \u003cem\u003eGeosmithia\u003c/em\u003e spp. and one of \u003cem\u003eOphiostoma novo-ulmi\u003c/em\u003e), obtained from the edges of actively growing fungal cultures, about 1 cm apart from each other near to the center of dish. Cultures were incubated in the dark at 24\u0026deg;C, and two radii of each colony on the growing edge opposite to the other fungus were measured after 48 h, 3, 5, and 8 days. Three plates per isolate were inoculated with two identical plugs as a control. Daily radial growth rates were compared by one-way ANOVA (Statistica 10, StatSoft Inc.).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cp\u003e1.2 Fungal metabolic potential analyses \u0026ndash; FF Plate\u0026trade;\u003c/p\u003e\n\u003cp\u003eThe phenotypic analysis was achieved using OmniLog\u0026reg; ID System multiplate reader (Biolog Inc., Hayward, CA, United States). Phenotype microarray experiments were performed using FF Biolog MicroArray\u0026trade; plates to cover a broad spectrum of carbon substrates. Every microarray plate contains 95 low-molecular-weight carbon sources from biochemical groups such as: amines/amides (n\u0026thinsp;=\u0026thinsp;6), amino acids (n\u0026thinsp;=\u0026thinsp;13), carbohydrates (n\u0026thinsp;=\u0026thinsp;44), carboxylic acids (n\u0026thinsp;=\u0026thinsp;17), polymers (n\u0026thinsp;=\u0026thinsp;5) and miscellaneous/other (n\u0026thinsp;=\u0026thinsp;10) [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. Each plate also contains water as a negative control (n\u0026thinsp;=\u0026thinsp;1). FF plates were stored at 4\u0026deg;C until needed.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeosmithia pumila\u003c/em\u003e (IVV7) and \u003cem\u003eO. novo-ulmi\u003c/em\u003e (ONU EAN H328), were selected for this trial since they are the most representative isolates of the species within the DED pathosystem, have been studied most extensively in recent years [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThree different phenotypic analyses were performed by inoculating the plates by i) \u003cem\u003eO. novo-ulmi\u003c/em\u003e; ii) \u003cem\u003eG. pumila\u003c/em\u003e; and their co-inoculum iii) \u003cem\u003eO. novo-ulmi\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eG. pumila.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBefore inoculation, fungi were cultivated on MEA for at least one week at 25\u0026deg;C in the dark to obtain an adequate amount of fungal material from the mycelial growth phase. The superficial layer of the mycelial mat was scraped with a sterile cotton swab to transfer fungal biomass from the colonies (a mixture of spores and mycelial cells) into sterile glass tubes containing 12 ml of an inoculating fluid provided by Biolog. A tissue grinder (Kontes\u0026reg; Duall\u0026reg; 21, Kimble Chase) was used to gently disrupt the fungal biomass to make the inoculum more homogenous. The same suspensions were used to prepare both the single and the co-inoculum. The transmittance of each inoculum was adjusted to 75% by adding more fungal biomass or inoculation fluid as required. The co-inoculum consisted in a mixture of equal volumes of the single strains spores\u0026rsquo; suspensions (1:1 ratio), which resulted, as well, in a final optical transmission of 75%. Three replica plates were prepared for each of the three analyses: \u003cem\u003eO. novo-ulmi\u003c/em\u003e, \u003cem\u003eO. novo-ulmi\u003c/em\u003e and \u003cem\u003eG. pumila\u003c/em\u003e (double culture), and \u003cem\u003eG. pumila\u003c/em\u003e (monoculture). The inoculated plates were kept at 25\u0026deg;C in darkness in the OmniLog\u0026reg; ID System multiplate reader (Biolog Inc., Hayward, CA, United States).\u003c/p\u003e\n\u003cp\u003eFungal growth was spectrophotometrically measured as optical density (OD) at 595 nm for 5 days, every 15 min, by the built-in camera and saved as OmniLog units generated by the Biolog\u0026reg; OmniLog PM software. The OD values of each isolate on the different substrates were normalized against those in the control well [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. Data analysis for fungal strains\u0026rsquo; metabolic profiling was conducted using Kinetic and Parametric software (Biolog). Phenotypes were determined based on the area under the kinetic curve of dye formation [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Heatmaps dendrogram was performed to show the correlation between the three treatments in relation to the utilization of C-sources from the FF microplates analyses.\u003c/p\u003e\n\u003cp\u003eFurthermore, a comprehensive assessment of synergistic substrate utilization was conducted by measuring the difference between substrate consumption in co-cultures of \u003cem\u003eOnu\u003c/em\u003e and \u003cem\u003eG. pumila\u003c/em\u003e compared to their respective monocultures. Synergy was calculated as: synergy = ((ONU\u0026thinsp;+\u0026thinsp;\u003cem\u003eG. pumila\u003c/em\u003e) \u0026minus; (ONU monoculture\u0026thinsp;+\u0026thinsp;\u003cem\u003eG. pumila\u003c/em\u003e monoculture))/2, as used by [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e], where positive values indicate synergistic utilization (substrate consumption in co-culture exceeds the sum of individual monocultures) and negative values indicate negative interactions.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eDaily fungal radial growth rates were compared by one-way ANOVA (Statistica 10, StatSoft Inc.) with Duncan test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eFor the phenotypic data, an ANOVA was conducted to assess statistical differences among the groups. When the ANOVA indicated a significant effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Tukey\u0026rsquo;s Honest Significant Difference (HSD) post‑hoc test was applied to identify pairwise differences among the three inoculums. The statistical analyses were performed on raw data of absorbance values at 90 h. Then, the raw data were normalized to 0\u0026ndash;1 range to perform a principal component analysis (PCA). The was employed to assess the metabolic differentiation between the three inoculums. All analysis were performed using R version 4.4.2 (R Core Team, 2024).\u003c/p\u003e\n\u003cp\u003eEcological analyses\u003c/p\u003e\n\u003cp\u003eThe niche overlap indices (NOI) were calculated according to Lee and Magan [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e] and Wilson and Lindow [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. A NOI value of 0.9 or above indicates a high degree of niche overlap and a competitive disadvantage for the target fungus [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. We also calculated the \u003cem\u003eO. novo-ulmi\u003c/em\u003e and \u003cem\u003eGeosmithia\u0026rsquo;\u003c/em\u003es ability to compete with each other (fungal competitiveness, FC) based on the formula by Blumenstein et al. [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. If the value of this function is greater than 1.0, one fungus exhibits competitive superiority relative to the other.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eFungal Growth Rate in Dual Culture on agar/water\u003c/h2\u003e\n\u003cp\u003eThe growth rate of \u003cem\u003eO. novo-ulmi\u003c/em\u003e strains was generally higher in dual culture with \u003cem\u003eGeosmithia\u003c/em\u003e spp. strains compared to monoculture controls (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Across all dual culture tests, \u003cem\u003eOphiostoma\u003c/em\u003e strains showed significantly higher growth rates when grown in dual culture with each \u003cem\u003eGeosmithia\u003c/em\u003e isolate tested, (Duncan test, p\u0026thinsp;\u0026le;\u0026thinsp;0.05), despite growth occurred on nutritionally inert substrate (water agar). In particular, Onu EAN (H328) grows significantly more with all \u003cem\u003eGeosmithia\u003c/em\u003e isolates tested (significant Duncan test, p\u0026thinsp;\u0026le;\u0026thinsp;0.05). No significant differences in growth rates were observed between \u003cem\u003eGeosmithia\u003c/em\u003e species when grown with \u003cem\u003eOphiostoma\u003c/em\u003e, apart from a few isolate-specific interactions, as shown in the table. Within each species, all strains grew at the same rate (non-significant Duncan test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eFungal growth rates in dual culture assays. Grey background indicates \u003cem\u003eO. novo-ulmi\u003c/em\u003e growth in the presence of \u003cem\u003eGeosmithia\u003c/em\u003e spp., while white background indicates \u003cem\u003eGeosmithia\u003c/em\u003e growth in the presence of \u003cem\u003eO. novo-ulmi\u003c/em\u003e strains. (+) indicates significantly increased growth compared to controls (Duncan's test, p\u0026thinsp;\u0026le;\u0026thinsp;0.05); (\u0026minus;) indicates significantly decreased growth compared to controls (Duncan's test, p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eFungal growth rate\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eG. pumila\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"7\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eG. funiculosa\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eG. langdonii\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"6\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eG. omnicola\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eG. ulmacea\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eG. flava\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eG. putterillii\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCNR39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIVV7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCNR102\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCNR48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eCNR31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCNR26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eCNR105\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eCNR8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eCNR32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eCNR23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCNR120\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCCF3342\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eOnu NAN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH172\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e182E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOPH123\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOPH32A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eOnu EAN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCTK11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH327\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH328\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eBiolog Results Analysis\u003c/h3\u003e\n\u003cp\u003eThe three phenotypic analyses carbon substrate utilization were compared revealing distinct metabolic patterns among the 95 substrates. Substrate activation was analyzed by quantifying the number of substrates with metabolic activity (OD\u0026thinsp;\u0026gt;\u0026thinsp;10 units) at each timepoint up to 90 hours.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThree distinct temporal phases characterized the metabolic dynamics of the two fungal species and their co-culture. During the early phase (12\u0026ndash;24 hours), \u003cem\u003eG. pumila\u003c/em\u003e initiated substrate utilization slowly, with only 13 substrates at 12 hours, progressively increasing to 50 by 24 hours. \u003cem\u003eO. novo-ulmi\u003c/em\u003e demonstrated a more rapid metabolic onset, utilizing 46 substrates at 12 hours and reaching 70 by 24 hours. The co-culture of \u003cem\u003eO. novo-ulmi\u003c/em\u003e \u0026amp; \u003cem\u003eG. pumila\u003c/em\u003e displayed intermediate metabolic activity, with 18 substrates at 12 hours and 63 substrates by 24 hours (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). During the intermediate phase (36\u0026ndash;48 hours), \u003cem\u003eG. pumila\u003c/em\u003e accelerated its expansion, while \u003cem\u003eO. novo-ulmi\u003c/em\u003e stabilised a plateau. The co-culture matched \u003cem\u003eG. pumila\u003c/em\u003e performance. During the late phase (60\u0026ndash;90 hours), \u003cem\u003eG. pumila\u003c/em\u003e and the co-colture continued its expansion (up to 92 and 88 substrates, respectively), while \u003cem\u003eO. novo-ulmi\u003c/em\u003e remained stable (77 substrates). Analysis of hourly substrate consumption rates (OD mean/hours) revealed distinct metabolic dynamics among the three treatments. \u003cem\u003eO. novo-ulmi\u003c/em\u003e exhibited a pronounced peak of approximately 4.2 at 24 hours then decreased rapidly after, indicating intense but transient metabolic activity. In contrast, \u003cem\u003eG. pumila\u003c/em\u003e and the combined culture maintained relatively stable and lower consumption rates (2.0\u0026ndash;2.5) throughout the observation period. After 24\u0026ndash;36 hours, all treatments showed a gradual decline in consumption rates, converging toward 1.5\u0026ndash;1.8 by 90 hours, suggesting a shift from active substrate utilization to a stationary metabolic phase (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Regarding average carbon substrate utilization, \u003cem\u003eG. pumila\u003c/em\u003e demonstrated the highest metabolic capacity, achieving approximately 155 OD units by 90 hours despite a slower initial growth phase. Conversely, \u003cem\u003eO. novo-ulmi\u003c/em\u003e exhibited rapid substrate utilization during the first 48 hours but subsequently plateaued at lower levels than the other treatments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eAnalysis of individual substrate categories revealed that the \u003cem\u003eG. pumila\u003c/em\u003e strain demonstrated exclusive or superior utilization of complex nitrogen substrates (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). N-Acetyl-L-Glutamic Acid, L-Pyroglutamic Acid, N-Acetyl-D-Galactosamine and Sebacic Acid were completely unused by \u003cem\u003eO. novo-ulmi\u003c/em\u003e but fully utilized in \u003cem\u003eG. pumila\u003c/em\u003e treatments and robustly exploited in co-culture (OD 90\u0026ndash;165). The use of L-phenylalanine showed a marked rise in co-culture, indicating an increase in the catabolism of aromatic amino acids with greater metabolic diversity. In contrast, the utilization of N-acetyl-D-mannosamine and Lactulose was exploited by both monocultures but not by the co-culture, suggesting direct metabolic antagonism or enzymatic competition.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNucleosides and Nucleotides.\u003c/em\u003e Adenosine showed the most striking synergistic response, remaining nearly unused by ONU in monoculture (OD\u0026thinsp;~\u0026thinsp;10\u0026ndash;25) yet activating to high levels in co-culture (OD 120\u0026ndash;195 by 48 h), demonstrating direct metabolic facilitation of the co-culture. Similarly, Uridine showed increased utilization level in co-culture, indicating a \u003cem\u003eG. pumila\u003c/em\u003e -mediated improvement.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOrganic Acids.\u003c/em\u003e Several carboxylic acids were exclusively or preferentially used by \u003cem\u003eG. pumila\u003c/em\u003e. Bromosuccinic Acid, p-Hydroxyphenylacetic Acid, and D-Saccharic Acid remained unexploited by \u003cem\u003eO. novo-ulmi\u003c/em\u003e alone but became metabolically available in dual culture.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePolyols and Sugar Alcohols\u003c/em\u003e. \u003cem\u003eO. novo-ulmi\u003c/em\u003e showed preferential utilization of polyols in monoculture, which were antagonistically suppressed in dual culture. D-Mannitol, L-Sorbose, Xylitol, Adonitol and Maltitol all exhibited marked suppression in co-culture (OD\u0026thinsp;~\u0026thinsp;50\u0026ndash;100 vs. 150\u0026ndash;200 in \u003cem\u003eO. novo-ulmi\u003c/em\u003e monoculture), suggesting competitive exclusion by \u003cem\u003eG. pumila\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eG. pumila\u003c/em\u003e enhanced the utilization of atypical monosaccharides. D-Ribose, D-Tagatose, D-Gluconic Acid, and Stachyose were scarcely used by \u003cem\u003eO. novo-ulmi\u003c/em\u003e, while they reached high levels of utilisation in dual culture and \u003cem\u003eG. pumila\u003c/em\u003e monoculture. Conversely, D-Arabinose and L-Fucose remained poorly utilized across all treatments, suggesting limited enzymatic capacity in both species.\u003c/p\u003e\n\u003cp\u003eCyclodextrin metabolism was \u003cem\u003eG. pumila\u003c/em\u003e -exclusive: \u0026alpha;-Cyclodextrin and \u0026beta;-Cyclodextrin were metabolized just by \u003cem\u003eG. pumila\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eSedoheptulosan utilization was absent in \u003cem\u003eO. novo-ulmi\u003c/em\u003e (OD\u0026thinsp;=\u0026thinsp;0) but dramatically increased in co-culture with \u003cem\u003eG. pumila\u003c/em\u003e (OD\u0026thinsp;=\u0026thinsp;95.58 vs. 10.39 \u003cem\u003eG. pumila\u003c/em\u003e monoculture), indicating synergistic metabolic activation.\u003c/p\u003e\n\u003cp\u003eComprehensive analysis of 95 distinct chemical substrates revealed substantial heterogeneity in synergistic responses (Table S2). The most pronounced positive synergistic responses were observed for carbohydrate-derived compounds and sugar alcohols. Sedoheptulosan exhibited the strongest synergy (+\u0026thinsp;90.39), representing a 90-unit increase in net substrate utilization in co-culture. Other highly synergistic substrates included \u0026alpha;-methyl-D-glucoside (+\u0026thinsp;84.50), N-acetyl-L-glutamic acid (+\u0026thinsp;84.30), and L-pyroglutamic acid (+\u0026thinsp;80.88). Conversely, several substrates exhibited strong negative synergy, indicating competitive inhibition or reduced substrate accessibility under co-culture conditions. Xylitol displayed the strongest antagonistic response (\u0026minus;\u0026thinsp;82.41), followed by D-glucosamine (\u0026minus;\u0026thinsp;45.62) and maltitol (\u0026minus;\u0026thinsp;44.63). Additional substantially antagonistic substrates included D-psicose (\u0026minus;\u0026thinsp;40.63), succinic acid (\u0026minus;\u0026thinsp;35.83), and salicin (\u0026minus;\u0026thinsp;33.26). All those difference are statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003ePCA Biplot Analysis of Metabolic Differentiation\u003c/h3\u003e\n\u003cp\u003ePrincipal component analysis of substrate utilisation patterns revealed distinct metabolic separation among \u003cem\u003eO. novo-ulmi\u003c/em\u003e, \u003cem\u003eG. pumila\u003c/em\u003e, and their co-culture (\u003cem\u003eO. novo-ulmi\u003c/em\u003e \u0026amp; \u003cem\u003eG. pumila\u003c/em\u003e), with the first two dimensions explaining 99.6% of total variance (PC1: 67.4%, PC2: 32.2%) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The biplot demonstrated clear spatial clustering of the three treatments, reflecting fundamental differences in substrate preference and enzymatic capacity. Onu occupied the upper-right quadrant, characterised by strong associations with polyols (D-mannitol, xylitol, adonitol, maltitol) and simple carbohydrates. In contrast, \u003cem\u003eG. pumila\u003c/em\u003e positioned in the upper-left quadrant, showing preferential utilisation of nitrogen-enriched substrates (L-pyroglutamic acid, N-acetyl-L-glutamic acid, N-acetyl-D-galactosamine), complex organic acids (bromosuccinic acid, p-hydroxyphenylacetic acid), nucleosides (adenosine, uridine), and exclusive utilisation of cyclodextrins (\u0026alpha; and \u0026beta;), and were completely unexploited by Onu. The co-culture occupied an intermediate central position, reflecting a metabolically integrated system rather than dominance by either partner. Compounds contributing most significantly to PC1 included N-acetyl D-glucosamine, salicin, and L-ornithine, indicating carbohydrate and amino acid metabolism as primary discriminatory factors. Compounds clustering along PC2 included various hexoses, pentoses, and organic acids, distinguishing strains based on specific metabolic pathways for sugar utilization and organic acid production.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eEcological analyses\u003c/h2\u003e\n\u003cp\u003eThe analysis of the nutritional profile of the three fungal strains revealed significant differences in their substrate utilization capacity. Niche overlap index (NOI) analysis revealed moderate nutritional overlap among the strains: \u003cem\u003eO. novo-ulmi\u003c/em\u003e shared 74% of its substrates with \u003cem\u003eG. pumila\u003c/em\u003e, while \u003cem\u003eG. pumila\u003c/em\u003e exhibited 82% overlap with \u003cem\u003eOphiostoma\u003c/em\u003e's substrates. These results suggest that the two fungi occupy partially overlapping but distinct nutritional niches.\u003c/p\u003e\n\u003cp\u003eRegarding fungal competitiveness (FC) according to the Blumenstein et al. [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e] model, \u003cem\u003eO. novo-ulmi\u003c/em\u003e demonstrated an FC of 0.89 against \u003cem\u003eG. pumila\u003c/em\u003e, indicating moderate competitive capacity for nutritional resources. In contrast, \u003cem\u003eG. pumila\u003c/em\u003e showed an FC of 1.15 against \u003cem\u003eO. novo-ulm\u003c/em\u003ei, suggesting superior competitiveness in the context of nutritional competition. These findings indicate that, although there is substantial metabolic overlap among the strains, \u003cem\u003eG. pumila\u003c/em\u003e possesses a greater capacity to compete for shared nutritional substrates.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe increased growth of \u003cem\u003eO. novo-ulmi\u003c/em\u003e in dual culture with \u003cem\u003eGeosmithia\u003c/em\u003e spp. on nutritionally inert water agar substrate presents an unexpected result; despite the competitive superiority of \u003cem\u003eGeosmithia\u003c/em\u003e (FC\u0026thinsp;=\u0026thinsp;1.15 versus 0.89), \u003cem\u003eO. novo-ulmi\u003c/em\u003e shows greater growth when physically close to \u003cem\u003eGeosmithia\u003c/em\u003e than in monoculture controls. This observation is consistent with its ecological specialization as a wood-colonizing pathogen that depends on complex organic substrates [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The absence of significant reciprocal growth enhancement for \u003cem\u003eGeosmithia\u003c/em\u003e species in co-culture, combined with the intermediate FC values, suggests an asymmetric mutualism in which \u003cem\u003eO. novo-ulmi\u003c/em\u003e benefits from physical proximity and possibly biochemical facilitation from the presence of the other fungus. The data indicate that physical contact between the two fungi establishes a localized metabolic interface through which selective nutrient exchange and chemical signaling occur [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Although phenotypic analysis shows that \u003cem\u003eG. pumila\u003c/em\u003e utilises more substrates than \u003cem\u003eO. novo-ulmi\u003c/em\u003e (93.68% versus 83.16% within 90 hours), the growth of \u003cem\u003eO. novo-ulmi\u003c/em\u003e in dual culture on nutrient-free substrate indicates that facilitation operates through non-nutritional mechanisms rather than nutrient supply. This is consistent with the current understanding that microbial coexistence often depends on chemical interactions and metabolic plasticity rather than simple competition for resources [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The growth enhancement observed on nutrient-depleted substrate suggests that \u003cem\u003eGeosmithia\u003c/em\u003e spp. may facilitate \u003cem\u003eO. novo-ulmi\u003c/em\u003e through non-nutritional mechanisms, including modulation of the local microenvironment, rather than through provision of exogenous nutrients [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe phenotypic analysis provides mechanistic insight into how this metabolic interaction operates. Although our study was focused to two fungal isolates, these strains were selected based on their extensive characterisation in previous studies, including documented events of HGT [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe two fungal species exhibited distinct metabolic strategies. \u003cem\u003eO. novo-ulmi\u003c/em\u003e demonstrated rapid utilisation of a greater number of substrates in the early phase (12\u0026ndash;24 hrs), while \u003cem\u003eG. pumila\u003c/em\u003e showed colonisation on fewer substrates, followed by accelerated expansion until reaching the highest number of activated substrates, at the late stage (60\u0026ndash;90 hrs). This difference in metabolic strategy can be explained by the nature of the fungal pathogens. \u003cem\u003eO. novo-ulmi\u003c/em\u003e employs a rapid-exploitation strategy on preferred substrates, maximising fitness within a narrow time window, presumably to maintain a high level of pathogenicity towards the host plant, a pattern also observed in soil pathogens such as \u003cem\u003ePythium aphanidermatum\u003c/em\u003e and \u003cem\u003eFusarium oxysporum\u003c/em\u003e f.sp. \u003cem\u003eradicis-lycopersic\u003c/em\u003ei [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In contrast, \u003cem\u003eG. pumila\u003c/em\u003e appears to adopt a more conservative metabolic strategy characterised by a progressive capacity to utilise the substrate throughout the observation period. Co-culture displays an intermediate metabolic profile that progressively converges toward the \u003cem\u003eG. pumila\u003c/em\u003e phenotype, exemplifying the well-established principle that metabolically superior competitors eclipse early colonizers in microbial communities [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe metabolic synergy, as utilisation of adenosine, is a widespread phenomenon in microbial communities [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. \u003cem\u003eO. novo-ulmi\u003c/em\u003e shows minimal adenosine metabolism in monoculture (OD\u0026thinsp;~\u0026thinsp;10\u0026ndash;25 at 48 hours) but achieves robust utilisation in co-culture (OD 120\u0026ndash;195 at 48 hours), with a 5\u0026ndash;19-fold increase. We observe a similar trend for adenosine 5'-monophosphate substrate. We hypothesize that this facilitation does not result from competitive inhibition, as \u003cem\u003eG. pumila\u003c/em\u003e effectively metabolises adenosines independently. Rather, the data indicate that \u003cem\u003eG. pumila\u003c/em\u003e could enzymatically degrade adenosine into other degradation products accessible to the metabolism of \u003cem\u003eO. novo-ulmi\u003c/em\u003e, as reported for other fungi [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eO. novo-ulmi\u003c/em\u003e showed a preferential utilisation of polyols (D-mannitol, L-sorbose, xylitol, adonitol, maltitol) in monoculture, with OD values ranging from 150 to 200. In dual culture, the utilisation of these substrates was significantly suppressed (OD\u0026thinsp;~\u0026thinsp;50\u0026ndash;100), suggesting competitive exclusion by \u003cem\u003eG. pumila\u003c/em\u003e. Similarly, N-acetyl-D-mannosamine was preferentially utilised by \u003cem\u003eO. novo-ulmi\u003c/em\u003e alone but antagonistically suppressed in co-culture. The increased growth of \u003cem\u003eO. novo-ulmi\u003c/em\u003e on nutrient-depleted agar despite the suppression of polyols indicates that growth facilitation does not depend on the utilisation of these compounds. Instead, \u003cem\u003eO. novo-ulmi\u003c/em\u003e appears to shift its metabolic investment towards alternative responses, such as the activation of stress pathways or the mobilisation of internal reserves, independent of external nutrients. This metabolic flexibility is consistent with documented stress responses in pathogenic fungi [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These findings reveal an interaction in which \u003cem\u003eG. pumila\u003c/em\u003e competitively monopolises specific nutritional niches (polyols, certain amino sugars) while simultaneously facilitating \u003cem\u003eO. novo-ulmi\u003c/em\u003e's access to alternative and metabolically distinct substrates.\u003c/p\u003e \u003cp\u003eAnalysis of the niche overlap index (NOI) quantified this division: \u003cem\u003eO. novo-ulmi\u003c/em\u003e shared 75.0% of its substrates with \u003cem\u003eG. pumila\u003c/em\u003e, while the latter showed a 94% overlap with \u003cem\u003eO. novo-ulmi\u003c/em\u003e substrates, an asymmetry that reflected \u003cem\u003eGeosmithia\u003c/em\u003e's broader metabolic capacity. These results, combined with some observed metabolic separation, suggest that the \u003cem\u003eUlmus\u003c/em\u003e-\u003cem\u003eO. novo-ulmi\u003c/em\u003e-\u003cem\u003eGeosmithia\u003c/em\u003e system is consistent with niche construction theory, according to which symbiotic partners construct niches through their metabolism and activities, creating a division that reflects both competitive and facilitating dynamics.[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrincipal component analysis confirmed this niche differentiation, demonstrating a clear metabolic separation (PC1: 67.4%, PC2: 32.2%) in which \u003cem\u003eO. novo-ulmi\u003c/em\u003e clustered with metabolic nodes associated with polyols, while \u003cem\u003eGeosmithia\u003c/em\u003e associated with nitrogen-enriched substrates and nucleosides. The co-culture occupied an intermediate position, tending towards the \u003cem\u003eG. pumila\u003c/em\u003e phenotype.\u003c/p\u003e \u003cp\u003eFungal competitiveness (FC) analysis revealed that \u003cem\u003eGeosmithia\u003c/em\u003e (FC\u0026thinsp;=\u0026thinsp;1.147) has a higher competitive capacity for shared nutritional resources than \u003cem\u003eO. novo-ulmi\u003c/em\u003e (FC\u0026thinsp;=\u0026thinsp;0.636), in line with field observations where \u003cem\u003eGeosmithia\u003c/em\u003e often dominates in ecologically competitive contexts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, this nutritional competitiveness becomes functionally irrelevant on nutrient-poor substrates such as water/agar, where exogenous carbon and nitrogen are absent. This context dependence suggests that on nutrient-free substrate, \u003cem\u003eGeosmithia\u003c/em\u003e likely facilitates \u003cem\u003eO. novo-ulmi\u003c/em\u003e growth through non-nutritional mechanisms. Microbial interactions are regulated by multiple factors, including chemical, biological, physical, and genetic elements, with substrate and nutrient composition playing a key role in determining interaction patterns [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Furthermore, fungal pathogens raise local pH through ammonia production, improving stress tolerance and growth conditions [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, fungi subjected to osmotic stress accumulate protective compounds (glycerol, trehalose, proline) that help cells survive drying stress [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The co-presence of \u003cem\u003eGeosmithia\u003c/em\u003e spp. may enhance the ability of \u003cem\u003eO. novo-ulmi\u003c/em\u003e to produce these protective compounds, allowing for more efficient use of internal resources for growth. This is consistent with studies showing that in nutrient-poor environments, fungal coexistence is determined by non-nutritional factors such as pH modification and stress tolerance rather than direct competition [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe distinction between metabolic potential (as revealed by PM analysis with defined substrates) and in vitro growth on \u0026ldquo;poor\u0026rdquo; substrate is ecologically fundamental to understanding the Onu-\u003cem\u003eGeosmithia\u003c/em\u003e spp. interaction within the DED pathosystem. In naturally colonised host tissues, microhabitat heterogeneity creates zones with variable nutrient availability: fresh xylem tissue provides abundant simple carbohydrates [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] preferred by Onu, while beetle excrement and degraded tissue offer nitrogen-enriched resources [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] exploited by \u003cem\u003eGeosmithia\u003c/em\u003e spp.. It has been observed [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] that the growth of many species of \u003cem\u003eGeosmithia\u003c/em\u003e is favoured by the urea, uric acid, and in part, ammonia, which demonstrates their ability to recycle nitrogen, probably originating from beetle excrement. Since the concentration of nitrogen in beetle galleries greatly affects their vitality [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], the presence of \u003cem\u003eGeosmithia\u003c/em\u003e spp. for nitrogen recycling seems to be of great importance within the DED pathosystem.\u003c/p\u003e \u003cp\u003ePathogenic fungi, such as \u003cem\u003eO. novo-ulmi\u003c/em\u003e, rapidly regulate their metabolism in response to available resources [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This metabolic flexibility allows Onu to increase overall growth despite limited access to its preferred substrates. Results indicate that \u003cem\u003eGeosmithia\u003c/em\u003e species releases nitrogen-containing compounds and nucleosides into the co-culture, redirecting Onu's metabolism towards purine and pyrimidine metabolism, as reported by Chitty et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] for human fungal pathogens. This metabolic shift is documented in nutrient-depleted fungal cells as a strategy to sustain essential biosynthetic processes and energy production [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of this research provide novel insights about the \u003cem\u003eO. novo-ulmi\u003c/em\u003e-\u003cem\u003eGeosmithia\u003c/em\u003e interaction, changing the perspective on the mycoparasitic interpretation of the interaction, in which morphological penetration of the hyphae was understood exclusively as predatory antagonism [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This study suggests that physical contact may establish a finely regulated metabolic exchange and chemical signalling, while the evolutionary stability of horizontal gene transfer events between these fungi (\u003cem\u003ecerato-ulmin\u003c/em\u003e gene transfer; [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]) supports long-term functional integration between these fungal species.\u003c/p\u003e \u003cp\u003eThis biological integration, combined with our metabolic data, supports the hypothesis that the two fungi have converged towards a form of functional symbiosis within the DED pathosystem. The distinction between metabolic potential (revealed by phenotypic analysis with microarrays with defined substrates) and growth on nutrient-poor substrates is fundamental from an ecological point of view: in naturally colonised host tissues, microhabitat heterogeneity creates zones with variable nutrient availability, and the ability of fungi to dynamically partition niches, exploit resources at different times, and facilitate growth in the absence of nutrients likely significantly contributes to their ecological success. The metabolic advantages deriving from their interaction are manifold: each fungus facilitates the other's access to otherwise inaccessible nutrient sources, including through a possible reciprocal exchange of metabolites and enzymatic activities, and \u003cem\u003eO. novo-ulmi\u003c/em\u003e grows significantly more than in monoculture without affecting \u003cem\u003eGeosmithia\u003c/em\u003e spp. growth. Collectively, these results suggest that their fungal coexistence may represent a mutualistic strategy conferring selective advantages over solitary survival, thus explaining the evolution of their ecological and biological interaction.\u003c/p\u003e \u003cp\u003eFuture investigations will be useful to characterise secondary metabolite profiles, pH dynamics, and osmolite production during co-culture growth to identify specific non-nutritional mechanisms driving the growth facilitation of \u003cem\u003eO. novo-ulmi\u003c/em\u003e under nutrient-deficient conditions. Such investigations will deepen our understanding of fungal pathogenesis in the context of complex microbial communities and inform strategies for disease management in natural ecosystems.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics Approval\u003c/strong\u003e \u003cp\u003eNo ethical approval was required for this study as it did not involve the use of any vertebrate animals or endangered insects.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eWe acknowledge financial support under the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, project: \u0026ldquo;Ulmus glabra protection in Italian peninsula \u0026ndash; MONTANA\u0026rdquo;, CUP: B53D23012340006, published on 2.2.2022 by the Italian Ministry of University and Research (MUR), and funded by the European Union \u0026ndash; NextGenerationEU.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.L.P. (Conceptualization, Data curation, Investigation, Methodology, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Project administration), H.B. (Data curation, Formal analysis, Investigation, Writing \u0026ndash; review \u0026amp; editing), N.L. (Conceptualization, Writing \u0026ndash; review \u0026amp; editing), A.G. (Investigation, Methodology), F.P. (Methodology, Writing \u0026ndash; review \u0026amp; editing), A.S. (Conceptualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePepori AL, Bettini PP, Comparini C et al (2018) \u003cem\u003eGeosmithia\u003c/em\u003e-\u003cem\u003eOphiostoma\u003c/em\u003e: a new fungus-fungus association. 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Int J Med Microbiol 301:400\u0026ndash;407\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ophiostoma novo-ulmi, Geosmithia spp., metabolic interactions, Dutch elm disease, Biolog FF Plates; carbon substrate utilization; dual growth rate; phenotyping","lastPublishedDoi":"10.21203/rs.3.rs-8842177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8842177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDutch elm disease (DED), caused by the invasive vascular pathogen \u003cem\u003eOphiostoma novo-ulmi\u003c/em\u003e, one of the most devastating pandemics affecting elms. Within beetle galleries and on elm bark beetle vectors, \u003cem\u003eO. novo-ulmi\u003c/em\u003e co-occurs with fungi of the genus \u003cem\u003eGeosmithia\u003c/em\u003e, yet the functional significance of this association remains poorly understood. This study investigates metabolic interactions between \u003cem\u003eO. novo-ulmi\u003c/em\u003e and \u003cem\u003eGeosmithia\u003c/em\u003e spp. using in vitro dual-culture experiments and phenotype microarray analysis to elucidate ecological mechanisms potentially influencing disease development and vector ecology. Dual-culture assays on dH\u003csub\u003e2\u003c/sub\u003eO agar revealed that \u003cem\u003eO. novo-ulmi\u003c/em\u003e exhibited significantly enhanced radial growth rates when cultured in proximity to \u003cem\u003eGeosmithia\u003c/em\u003e isolates. Phenotypic microarray analysis revealed distinct metabolic strategies: \u003cem\u003eO. novo-ulmi\u003c/em\u003e utilised substrates within 12\u0026ndash;24 hours and preferred polyols and simple carbohydrates, while \u003cem\u003eG. pumila\u003c/em\u003e adopted a progressive colonisation strategy, ultimately utilising 92 substrates in 90 hours and demonstrating greater utilisation of nitrogen-enriched substrates, nucleosides and complex organic acids. Fungal competitiveness analysis indicated \u003cem\u003eG. pumila\u003c/em\u003e superiority, yet \u003cem\u003eO. novo-ulmi\u003c/em\u003e showed greater growth facilitation in co-culture, suggesting asymmetric mutualism through non-nutritional mechanisms. These results also suggest that physical proximity between \u003cem\u003eO. novo-ulmi\u003c/em\u003e and \u003cem\u003eGeosmithia\u003c/em\u003e spp. establishes a localized metabolic interface enabling chemical signaling and nutrient exchange. This mutualistic interaction, demonstrated by the increased growth of \u003cem\u003eO. novo-ulmi\u003c/em\u003e in co-culture without suppressing \u003cem\u003eGeosmithia\u003c/em\u003e spp., likely confers selective advantages that explain the evolutionary success of their ecological partnership. Within naturally colonised host tissues, such metabolic interaction may enhance pathogen establishment and persistence, with implications for disease epidemiology, beetle vector ecology, and potential disease management strategies.\u003c/p\u003e","manuscriptTitle":"Exploring metabolic interaction between Ophiostoma novo-ulmi and Geosmithia spp","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 14:29:18","doi":"10.21203/rs.3.rs-8842177/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-11T19:44:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-08T00:30:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T14:06:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-23T02:11:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214328751536417036005432278975179146896","date":"2026-02-18T06:56:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248914185385291774654257324440525200842","date":"2026-02-14T01:36:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275107489254191205285438937677757336391","date":"2026-02-11T16:36:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235503369081994822830810087842308738494","date":"2026-02-11T07:37:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-11T07:25:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-11T04:49:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-11T04:48:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Ecology","date":"2026-02-10T13:41:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ae69b2b1-4d4f-4e63-aa15-717edcc2a1b5","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T18:08:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 14:29:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8842177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8842177","identity":"rs-8842177","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}