Sesquiterpene Biosynthetic Gene vir4 from Trichoderma virens Enhances Direct Herbivore Resistance while Maintaining Indirect Defense

Sesquiterpene Biosynthetic Gene vir4 from Trichoderma virens Enhances Direct Herbivore Resistance while Maintaining Indirect Defense | 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 Sesquiterpene Biosynthetic Gene vir4 from Trichoderma virens Enhances Direct Herbivore Resistance while Maintaining Indirect Defense Noor Agha Nawakht, Artemio Mendoza-Mendoza, Michael Rostás This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6548157/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jan, 2026 Read the published version in Journal of Chemical Ecology → Version 1 posted 9 You are reading this latest preprint version Abstract Trichoderma species are widely used as root-colonizing biocontrol agents that enhance plant resistance to biotic and abiotic stresses while promoting growth. These fungi produce diverse volatile and non-volatile metabolites that mediate interactions with plants. Trichoderma can influence both direct and indirect plant defenses, including the release of herbivore-induced plant volatiles (HIPVs) that attract natural enemies of herbivores. In this study, we examined the effects of T. virens and its vir4 gene ( regulating terpenioid synthesis) knockout-mutant on maize ( Zea mays ), the herbivore Helicoverpa armigera , and its predator Macrolophus pygmaeus . Previous research has shown that T. virens differentially modulate maize root gene expression and specialized metabolite concentrations. Here, we found that caterpillars feeding on maize seedlings colonized by wild-type T. virens gained significantly less weight than those feeding on maize colonized by the vir4 knockout mutant or uncolonized plants, suggesting that the vir4 gene cluster contributes to herbivore resistance. Although fungal colonization led to moderate changes in HIPV composition, total volatile emissions remained unchanged. In Y-tube assays, M. pygmaeus preferred caterpillar-infested maize over healthy plants, but fungal colonization did not significantly affect predator behavior. Our findings demonstrate that T. virens enhances direct plant defense against herbivores while neutralising indirect defense through a mechanism regulated by terpenoid synthesis depending on vir4 gene. Further research is needed to elucidate the metabolic changes in maize induced by T. virens that contribute to reduced herbivore performance. Trichoderma virens Helicoverpa armigera Macrolophus pygmaeus maize tri-trophic interactions GC-MS HIPVs defense priming Figures Figure 1 Figure 2 Figure 3 INTRODUCTION The genus Trichoderma (teleomorph Hypocrea) is a globally distributed soil-borne fungus that thrives in diverse habitats (Woo et al. 2022 ). It plays a key role in sustainable agriculture, acting as a biopesticide, bio-stimulant, and plant growth promoter while enhancing plant defenses (Harman et al. 2004 ; Woo et al. 2022 ). As an opportunistic symbiont, Trichoderma colonizes the rhizosphere and the apoplast of plant roots, inhabiting intercellular spaces and outer cell layers (Harman et al. 2004 ; Hermosa et al. 2012 ; Brotman et al. 2013 ; Nogueira-Lopez et al. 2018 ). Traditionally, its biocontrol activity was attributed to mycoparasitism, antibiosis, and competition for space and resources (Harman 2005 ; Vinale et al. 2008 ). However, recent studies highlight its role in inducing systemic and localized plant defense responses (Nawrocka 2013 ; Poveda 2021 ; Monte 2023 ). Trichoderma spp. produce a diverse array of volatile and non-volatile secondary metabolites that contribute to their bioactivity and defense-inducing properties in host plants (Hermosa et al. 2014 ; Jiménez-Bremont et al. 2024 ; Reino et al. 2007 ; Salwan et al. 2019 ; Vinale & Sivasithamparam. 2020; Zeilinger et al. 2016 ). Colonization of maize roots by Trichoderma spp. can significantly alter the host plant’s metabolome (Vinale et al. 2008 ; Vinci et al. 2018 ), proteome, and transcriptome (Harman 2005 ; Marra et al. 2006 ; Alfano et al. 2007 ; Shoresh & Harman 2008 , Nogueira-Lopez et al. 2018 ). Schweiger et al. ( 2021 ) demonstrated that the wild-type T. virens strain Gv29.8 and its Δvir4 mutant, which is deficient in sesquiterpene biosynthesis, differentially modulate maize root gene expression and metabolic composition. Their study revealed that T. virens colonization profoundly affects several metabolites derived from the shikimate pathway, including flavonoids and other compounds, in a genotype-dependent manner. These metabolic changes may influence not only belowground plant-insect interactions but also aboveground interactions, as Trichoderma spp. can induce systemic resistance in leaves, potentially triggering cascading effects across trophic levels. Several studies have reported Trichoderma -induced resistance against herbivorous insects from different feeding guilds, including lepidopterans (Contreras-Cornejo et al. 2018 ; Coppola et al. 2019a ; Macías-Rodríguez et al. 2020 ). For example, T. atroviride reduced aphid and caterpillar fitness in maize and tomato by modulating plant defense mechanisms (Coppola et al. 2019b ). Similarly, T. harzianum negatively affected Nezara viridula feeding behavior in tomato by enhancing direct defense responses (Alinc et al. 2021). Plants communicate with their environment through volatile organic compounds (VOCs), which can mediate both above- and belowground interactions (Massalha et al. 2017 ; Thompson et al. 2023 ). Insect feeding, in particular, triggers the release of herbivore-induced plant volatiles (HIPVs), which play a critical role in plant defense by attracting natural enemies of herbivores (Aartsma et al. 2017 ; Hilker & Meiners. 2006; Dicke et al. 2009 ). Microbial colonization, including beneficial fungi, can influence the emission of HIPVs by modifying both the composition and quantity of emitted volatiles. These changes, in turn, may affect the behavior and effectiveness of natural enemies in locating their herbivorous prey (Holopainen & Gershenzon. 2010; Pangesti et al. 2013 ; Pineda et al. 2010 ; Fernandez-Conradi et al. 2018 ; Schausberger et al. 2012 ). For instance, Trichoderma harzianum T22 was shown to enhance VOC-mediated defenses by increasing the attraction of aphid parasitoids in tomato (Coppola et al. 2017 ). Furthermore, T. harzianum and T. atroviride strain IMI 206040 improve maize tolerance to herbivores by modifying the emission of volatile terpenes that attract predators (Contreras-Cornejo et al. 2018 , 2021 ). Similarly, T. harzianum primes tomato plants to emit VOCs that attract parasitoids of aphids and stink bugs (Coppola et al. 2017 ; Alinc et al. 2024). H. armigera (Lepidoptera: Noctuidae) is a polyphagous caterpillar that infests economically important crops, including maize ( Zea mays L.). Its high fecundity, broad host range, and facultative diapause contribute to its status as a major pest. The zoophytophagous mirid bug M. pygmaeus (Hemiptera: Miridae) is an efficient predator of various herbivorous pest insect including H. armigera and is attracted to HIPVs emitted by crops such as tomato and tobacco (Maselou et al. 2019 ; Ingegno et al. 2016 ; Urbaneja et al. 2009 ; Ebrahimi et al. 2019 ). However, no studies have examined M. pygmaeus attraction to maize HIPVs particularly in the presence of T. virens strains as endophytic root colonizers. Since T. virens and its vir4 knockout mutant influence maize root metabolic profile (Schweiger et al. 2021 ), we hypothesized that these changes could extend systemically to higher trophic levels, affecting herbivores and their natural enemies aboveground. Therefore, this study aimed to investigate: (i) how maize root colonization by both T. virens genotypes affects H. armigera survival and weight gain, (ii) whether fungal colonization alters maize VOC composition or quantity and (iii) the consequences of these changes for M. pygmaeus foraging behavior within this multitrophic system. MATERIALS AND METHODS Insects rearing. Eggs of H. armigera were supplied by Bayer AG Crop Sciences (Monheim, Germany), and the larvae were reared on an artificial diet. Eggs were placed in plastic containers with slices of the diet and kept in an environmental chamber at 23°C, 65 ± 5% relative humidity, and a 14-hour light/10-hour dark photoperiod to induce hatching. After hatching, the neonates were reared on artificial diet until they reached the desired developmental stage, after which they were used in experiments. Adults of M. pygmaeus were obtained from Katz Biotech AG (Germany), along with Sitotroga eggs as supplementary food. A colony was established in BugDorm insect-rearing cages (BugDorm®, MegaView Science Co., Ltd.Taiwan). Potato tubers were placed on moistened soil in a tray inside the cages, and Sitotroga eggs were provided in a small Petri dish. Male and female adults of M. pygmaeus were released into the cage for mating and reproduction. Emerging adults were continuously transferred to new cages, maintained at ambient room temperature. Fungal culture and inoculum preparation . The fungal strains were cultured, and inoculum was prepared as described by Schweiger et al. ( 2021 ). Two genotypes of the T. virens strain Gv29.8 were used in the study: the wild-type and the Δvir4 knockout mutant, which is defective in sesquiterpene biosynthesis, hereinafter referred to as “wild-type” and “mutant”, respectively. Both fungal strains were propagated on potato dextrose agar (PDA) (Carl Roth GmbH, Karlsruhe, Germany). Regular sub-culturing was done in sterile Petri dishes containing PDA, and the plates were kept in an environmental chamber under controlled conditions of 25 ± 2°C, 65% relative humidity, and a 12-hour light: 12-hour dark cycle for 7 days to promote conidiation. To harvest the conidia, fungal plates were flooded with 15–20 ml of sterile water using a pipette. A microscope slide was used to gently scrape the surface of the media plate to detach the conidia from the mycelium. Using a funnel, the resulting suspension was then filtered through a cheesecloth into an Erlenmeyer flask. The final concentration of conidia for each fungal strain was adjusted to 1x10 ^6 conidia/ml to be used in subsequent inoculation procedures. Plant material and fungal inoculation. Seeds of maize, hybrid line 34H31 (Pioneer® Brand Products, Gisborne, New Zealand), were used in the experiments, and the inoculation procedure followed Nogueira-Lopez et al. ( 2018 ). Prior to inoculation, the seeds were surface sterilized by submerging them in 2% sodium hypochlorite (NaOCl) solution, followed by 70% ethanol for 7 min each. The sterilization process was followed by five washes with autoclaved tap water. The success of sterilization was confirmed by plating 100 µL of water from the final rinse on PDA (Carl Roth GmbH, Karlsruhe, Germany) and incubating under the same conditions used for fungal culture preparation. Seeds were placed in Petri dishes with moistened sterilized-filter paper to pre-germinate overnight. The following day, seeds were inoculated with the fungal spore suspension individually at a conencetration of 1x10^ 6 conidia under clean bench. Three treatment groups were prepared: (1) seeds inoculated with T. virens wild-type strain, (2) seeds inoculated with the mutant strain, and (3) a control group where kernels were mock-inoculated with sterilized water using the same method as the fungal spore treatment. All procedures were conducted under sterile conditions to prevent contamination. Treated seeds were sown in plastic pots containing gamma-irradiated soil. The pots were maintained in climate-controlled chambers with 80% relative humidity, a 16:8 light-dark cycle, and a temperature of 25°C to allow for proper growth of the maize plants. Herbivore performance bioassay . To assess the impact of fungal colonization on herbivore survival and weight gain, 2nd instar larvae of H. armigera were weighed and randomly assigned to 7-days old maize seedlings. Each seedling received a single larva placed into the whorl. The total sample size was 24 seedlings per treatment group (control, wild-type, mutant). To prevent larval escape and ensure airflow, each potted seedling was covered with a perforated cellophane plastic bag. The plants were then placed in an environmental chamber with 25°C, a 16-hour light/8-hour dark photoperiod, and 80% relative humidity. The caterpillars were weighed again 3, 6, and 9 days post infestation (dpi)—to determine the weight gain across different treatments. Additionally, survival was monitored daily for each treatment group throughout the 9-day feeding period to track mortality rates. Collection and analyses of the volatile blend . To collect and analyze the VOCs emitted by maize seedlings treated with T. virens wild-type, mutant, or control, a push-pull dynamic headspace VOCs collection system was employed using the lower part of a six-arm olfactometer (Turlings et al. 2004 ). Maize seedlings were infested with ten 2nd instar larvae of H. armigera one day before volatile collection. The VOCs were collected using volatile trapping filters containing 30 mg of 80–100 mesh Porapak Q (Volatile Collection Trap LLC, FL, USA), attached to the outlet air pipe of the odor source vessels. A central in-house compressor provided humidified and filtered air at an inflow rate of 1 l/min. Air was pulled out from the odor source vessels at a flow rate of 0.8 l/min, using a membrane pump. The VOCs collection started daily at 10:00 a.m. and lasted for four hours. All equipment, including glassware and vessels, was thoroughly cleaned with demineralized water, 99.5% acetone, and oven-dried at 180°C for two hours to prevent contamination. The filters were washed with 1 ml of dichloromethane (DCM) before use as described (Rahman et al. 2024). After VOCs collection, the trapped VOCs were eluted from the filters by rinsing them with 150 µl of DCM into 1 ml glass vials. The samples were stored at -80°C for further analysis, with 200 ng of 1,2,3,4-tetrahydronaphthalene added as an internal standard. For analysis, 40 µl of the final solution was transferred to glass vials with glass inserts for the GC-MS autosampler. A 2 µl aliquot was injected in pulsed splitless mode into the GC-MS system (5977B HES MSD, Agilent Technologies). The GC oven temperature was initially set at 40°C for 3 min, then increased gradually at a rate of 8°C min⁻¹ to 320°C, which was held for 10 min. Helium was used as the carrier gas at a constant flux of 1.5 ml/min. Chromatograms were automatically integrated using Agilent MSD ChemStation software and mass spectra were compared to those in the libraries NIST17 and Wiley11 for tentative identification. For further confirmation of compound identities, the retention index (RI) of each compound was calculated and compared to the RI values listed in the Van Den Dool and Kratz RI Table available in the NIST Chemistry Webbook library. Quantification of the compounds was based on their mass peak areas relative to the peak area of the internal standard. This method allowed for relative comparisons of the amounts of each volatile emitted in the treatments. The experimental treatments consisted of the following setups: (1) maize, (2) maize infested with herbivores, (3) maize colonized by the wild-type and infested with herbivores, (4) maize colonized by the mutant and infested with herbivores, along with a blank (empty collection vessel to control for background contamination). Response of M. pygmaeus to Herbivore-Induced Maize Volatiles. Y-tube olfactometer assays were conducted to assess the response of M. pygmaeus to volatiles emitted by maize treated with fungi and/or herbivores. The Y-tube had an 8 mm inner diameter, a 20 cm length for both the entry and side arms, and a 70° angle between the side arms. The methods were adapted from Lins et al. ( 2014 ), Maselou et al. ( 2019 ), and Silva et al. ( 2021 ). Maize seedlings (treated with fungal strains or left untreated as control) were infested with ten 2nd instar H. armigera larvae one day before the experiment to induce the release of VOCs. Seedlings were enclosed in plastic bags sealed at both ends. Two plastic tubes were inserted into the bags: one to allow inlet air from the air pump and the other to connect to the arms of the Y-tube olfactometer. Humidified and charcoal-filtered air was provided by an air compressor (Stimulus Controller CS-55 V2, Ockenfels Syntech GmbH, Germany), with the airflow adjusted to 0.3 L min⁻¹. Adults of M. pygmaeus were starved for 18 h before the experiment. Each individual insect was introduced into the central arm of the olfactometer and observed for a maximum of 10 min to make a choice by moving into one of the arms connected to an odor source. Insects moving 10 cm past the branching point towards one of the odor sources were considered to have made a choice. Insects that did not select a side within the 10-min period were classified as unresponsive and were excluded from the analysis. Five adult female insects were tested per plant set, and after each test, the olfactometer and odor sources were replaced. The control treatment followed the same procedure but without herbivore infestation. Before use, all olfactometer components were cleaned with Contrad 70® detergent (Decon Laboratories Limited, VWR International, Hove, East Sussex, UK), rinsed with distilled water, and air-dried overnight at room temperature. Experiments were conducted at room temperature under a fume hood, and to avoid positional bias, the positions of the odor sources (infested vs. healthy) were alternated between the left and right side arms. To compare the preferences of M. pygmaeus for different volatile profiles, six treatment combinations were tested pairwise using the Y-tube olfactometer. The treatment comparisons were as follows: (1) maize vs. maize infested with herbivores (M vs. MH); (2) maize vs. maize inoculated with the wild-type (M vs. MW); (3) maize vs. maize inoculated with the vir4 mutant (M vs. MV); (4) maize infested with herbivores vs. maize inoculated with wild-type and infested with herbivores (MH vs. MWH); (5) maize infested with herbivores vs. maize inoculated with mutant and infested with herbivores (MH vs. MVH) (6) maize inoculated with wild-type and infested with herbivores vs. maize inoculated with mutant and infested with herbivores (MWH vs. MVH). Individuals of M. pygmaeus were tested only once, with a total of 30 insects per treatment pair. The positions of the odor sources connected to the olfactometer were exchanged after testing five insects, and seedlings were discarded after each test. Data analyses and statistical procedures . All statistical analyses and data visualizations were performed using R. A linear mixed-effects model was fitted to assess the effects of treatment, time, and their interaction on caterpillar weight gain. Treatment and time were treated as fixed effects, while individual caterpillars were considered random effects to account for repeated measures. Tukey’s Honestly Significant Difference (HSD) test, adjusted for multiple comparisons, was used for post-hoc mean comparisons. These analyses were conducted using the lme4 , car , and emmeans packages in R. The relevant statistical assumptions were thoroughly evaluated. Normality of residuals was assessed through graphical inspections and the Shapiro-Wilk test, while homoscedasticity was evaluated using Levene's test. Kaplan-Meier survival analysis, along with the log-rank test, was used to determine statistical differences in caterpillar survival across treatments. For the analysis of volatile emissions, the total volatile emission data were log-transformed to meet the assumptions of ANOVA and Tukey’s HSD test was conducted for mean separation. The quantities of individual VOCs among treatments were compared using the Kruskal-Wallis non-parametric test followed by Dunn test with a Bonferroni correction. Heatmap was generated using the pheatmap package in R and color gradients were customized using the RColorBrewer package. To test the null hypothesis of no preference by the predator between the two odor sources, a Chi-squared (χ²) test was performed. A significance threshold of p ≤ 0.05 was applied for all statistical tests. RESULTS Herbivore performance and survival . Larval performance was evaluated for each treatment by monitoring weight gain at various time points post-feeding. Statistically significant differences in fresh body mass were observed among caterpillars fed on different treatments (Fig. 1 A). A linear mixed-effects model revealed significant main effects for both treatment (F 2.64, 11 = 3.717, p = 0.02) and day (F 3, 177.31 = 159.68, p < 0.001), as well as a significant interaction between treatment and day (F 6, 177.31 = 3.026, p = 0.007). Further pairwise comparisons showed that by day 9, caterpillars feeding on maize seedlings colonized by the wild-type genotype had significantly lower body weights compared to those on both the control (p = 0.01) and mutant (p < 0.001) treatments (Fig. 1 A). On the other hand, no significant differences in caterpillar survival were observed among the treatments over the 9-days experimental period (Fig. 1 B) (Log-Rank test, χ² = 1.63, df = 2, P = 0.44). The experiments were terminated on day 9, as some seedlings in certain treatments (i.e., control or mutant) were fully consumed by the caterpillars towards the 9th day. VOCs emission. Herbivory induced a distinct volatile bouquet, with some qualitative differences attributed to the fungal strains. Comparing the wild-type and mutant treatments, certain compounds were detected in one treatment but absent in the other. For instance, ( Z )-3-hexen-1-yl acetate was not found in the wild-type treatment but was present in the mutant treatment. Additionally, α‑bergamotene was identified in both fungal treatments but was absent in the herbivore-only treatment (Table 1 ). However, the quantity of individual VOCs did not differ statistically among treatments. ANOVA revealed significant differences in total volatile emissions between treatments (F 3, 24 = 3.18, p = 0.04; Fig. 2 A). A Tukey post-hoc test indicated that healthy maize emitted significantly fewer volatiles compared to the mutant-inoculated treatment (p = 0.03), while differences with other treatments were not statistically significant. Only trace amounts of VOCs were released from control maize. A heatmap visualization (Fig. 2 B) illustrates the variation in volatile profiles across treatments, suggesting that fungal colonization may have led to qualitative changes in the plant's volatile profile, albeit to a limited extent. Notably, herbivore-infested maize treated with the fungal mutant exhibited the highest VOC emissions, including δ-cadinene, ( E )-4, 8-dimethylnona-1, 3,7-triene (DMNT), and ylangene, Similarly, the wild-type treatment also showed elevated levels of certain VOCs, such as delta-cadinene, DMNT, and farnesene, although in lower quantities compared to the mutant treatment. Hierarchical clustering from the heatmap indicates that the treatments colonized by fungal strains and subjected to herbivory shared a somewhat similar volatile profile, as reflected by their clustering together. Furthermore, the principal component analysis (PCA) score plot (Fig. 2 C) shows clear separation between the treatments. PC1 explains 45.29% of the total variance, while PC2 accounts for 27.16%, together explaining 72.45% of the total variance. Particularly, the overlap between the mutant-herbivory and wild-type-herbivory treatments suggests that both treatments induce similar, yet slightly distinct, VOC responses, indicating their potential role in modulating the maize VOCs profile. Note, that untreated maize was not included in the analysis as most compounds were absent. Table 1 Estimated total VOC emission rate (ng/h/plant) from maize seedlings under different treatments: M = uninfested control maize, MH = maize infested with H. armigera caterpillars, MVH = maize inoculated with the mutant and infested with caterpillars, and MWH = maize inoculated with the wild-type and infested with caterpillars. Single VOCs were compared among treatments using the Kruskal-Wallis non-parametric test. Values are presented as mean ± standard error (SE), n = 7. Value in the same row sharing the same letters are not statistically different at p ≤ 0.05. n.d.= not detected. Compound Total VOCs emission ng/h/plant ± SE p value M MH MVH MWH (Z)-3-Hexen-1-yl acetate n.d. 5.97 ± 5.97 a 4.74 ± 2.27 a n.d. > 0.05 DMNT n.d. 3.33 ± 1.36 a 12.71 ± 5.10 a 4.00 ± 1.70 a > 0.05 Indole n.d. 2.26 ± 0.82 a 11.12 ± 6.19 a 4.90 ± 1.70 a > 0.05 Cyclosativene 0.37 ± 0.25 a 1.02 ± 0.44 a 1.60 ± 0.54 a 1.23 ± 0.60 a > 0.05 Ylangene 0.26 ± 0.19 a 0.60 ± 0.33 a 1.02 ± 0.33 a 0.69 ± 0.38 a > 0.05 α-Bergamotene n.d. n.d. 0.08 ± 0.08 a 0.83 ± 0.08 a > 0.05 β-Farnesene n.d. 0.21 ± 0.11 a 0.74 ± 0.33 a 0.36 ± 0.17 a > 0.05 δ-Cadinene n.d. 0.06 ± 0.06 a 0.05 ± 0.05 a 0.14 ± 0.14 a > 0.05 Olfactometer response of the predator. The response of M. pygmaeus to volatile blends released by maize seedlings colonized by different fungal strains, with or without herbivory, was assessed using a Y-tube olfactometer. The choices made by the predator, represented as percentages, indicate their preference for one arm of the olfactometer over the other. Predators significantly preferred the arm containing herbivore-damaged maize seedlings as the odor source compared to the control (Fig. 3 ). When fungal inoculation was also involved, the predators exhibited a significant preference for herbivore-infested maize seedlings over uninfested controls, regardless of fungal inoculation (χ² = 4.17, df = 1, P = 0.04) (Fig. 3 ). However, Chi-squared tests indicated no significant preference for volatile blends associated with different fungal treatments used in different combinations. Specifically, predator preference did not differ significantly between maize inoculated with the wild-type and infested with caterpillars (MWH) and maize inoculated with the mutant and infested with caterpillars (MVH) (χ² = 1.09, df = 1, P = 0.29). Similarly, no significant differences were observed between uninfested control maize (M) and wild-type-inoculated maize (MW) (χ² = 0.20, df = 1, P = 0.65); uninfested control maize (M) and mutant-inoculated maize (MV) (χ² = 0.40, df = 1, P = 0.82); herbivore-infested maize (MH) and wild-type-inoculated, herbivore-infested maize (MWH) (χ² = 0.36, df = 1, P = 0.54); or herbivore-infested maize (MH) and mutant-inoculated, herbivore-infested maize (MVH) (χ² = 0.04, df = 1, P = 0.83). DISCUSSION Trichoderma spp. are widely recognized as effective biocontrol agents against phytopathogens, yet their role in mediating plant-insect interactions remains underexplored (Poveda 2021 ; Lelio et al. 2023 ). Building on the findings of Schweiger et al. ( 2021 ), which demonstrated that colonization of maize roots by wild-type and mutant T. virens differentially alters the plant root metabolome, this study investigated whether these metabolic changes influence the development and survival of H. armigera caterpillars aboveground. Additionally, we assessed the effects of T. virens colonization on indirect defense mechanisms involving the predator M. pygmaeus . Our results demonstrate that maize colonized by wild-type T. virens significantly inhibited H. armigera caterpillar development compared to mutant-colonized and control plants, suggesting that T. virens enhances direct systemic resistance in maize. This effect is likely mediated by fungal sesquiterpenes encoded by the vir4 gene cluster, underscoring their importance in plant defense. Induced systemic resistance is a key defense mechanism in plants, wherein beneficial rhizosphere microbes, such as Trichoderma spp., prime plants for enhanced defense against pathogens and herbivores (Pieterse et al. 2014 ). Trichoderma spp. produce a diverse array of bioactive secondary metabolites, including sesquiterpenes, which play critical roles in plant-microbe and plant-insect interactions (Kramer & Abraham 2012 ; Vinale et al. 2008 ; Reino et al. 2007 ). These metabolites act as signaling molecules and priming agents, systemically enhancing plant defense responses when released into the rhizosphere (Contreras-Cornejo et al. 2016 ; Shoresh et al. 2010 ; Pieterse et al. 2014 ). The poor performance of H. armigera on maize colonized by wild-type T. virens may thus be attributed to the priming or induction of plant defenses by sesquiterpenes encoded by the vir4 gene cluster. This aligns with previous reports of Trichoderma -mediated host defense priming in various crops against herbivores across different feeding guilds (Alınç et al. 2021 ; Coppola et al. 2019b ; Morán-Diez et al. 2021 ). However, also direct deterrence of the herbivore by vir4 -derived metabolites that may be translocated from root to shoot cannot be ruled out and merits further investigation. Our findings are align with previous research on Trichoderma -plant-insect interactions. For instance, T. gamsii altered the foliar metabolome of Arabidopsis thaliana , reducing weight gain in Trichoplusia ni (Zhou et al. 2018 ). Similarly, T. atroviride colonization limited herbivory by Spodoptera frugiperda through the induction of plant resistance, which was partly mediated by fungal-derived antifeedant secondary metabolites (Contreras-Cornejo et al. 2018 ). It also negatively impacted the development of S. littoralis and aphids via transcriptional changes in defense-related genes (Coppola et al. 2019b ). Inoculation with T. harzianum reduced the growth rate of Nezara viridula by enhancing direct defense mechanisms and priming tomato plants (Alınç et al. 2021 ) while also enhancing maize root resistance to Phyllophaga vetula through the antifeedant activity of fungal-derived 6-pentyl-2H-pyran-2-one (6-PP) (Contreras-Cornejo et al. 2021 ). Additionally, T. afroharzianum and T. atroviride have been shown to enhance tomato resistance against S. littoralis and M. euphorbiae (Lelio et al. 2022, 2023 ). Collectively, these findings underscore the potential of T. virens in enhancing maize direct systemic resistance against H. armigera . Although no direct mortality of H. armigera caterpillars was observed in our study, the relatively short experimental period (9 days) may have limited the detection of longer-term effects. Maize responds to herbivory by releasing a blend of volatiles that attract natural enemies and prime neighboring plants for defense (Turlings & Ton 2006 ; Kim & Felton 2013 ; Thompson et al. 2023 ). Beneficial fungal colonization can influence HIPV emissions (Holopainen & Gershenzon 2010 ; Pangesti et al. 2013 ; Fernandez-Conradi et al. 2018 ), potentially shaping interactions at higher trophic levels. We investigated whether T. virens colonization alters maize HIPV emissions induced by H. armigera feeding. Our study identified a range of volatiles, including monoterpenes, sesquiterpenes, aromatic compounds, and green leaf volatiles (GLVs), primarily released in response to herbivory. However, T. virens root colonization had little influence on the composition of HIPVs emitted by maize leaves. Despite some variability in individual compounds such as ( Z )-3-hexen-1-ol acetate, neither the wild-type nor the vir4 mutant significantly affected the overall HIPV blend. In contrast, fungal-induced changes in VOC emissions and indirect plant defenses has been documented in other systems. For example, T. harzianum enhanced methyl salicylate and β-caryophyllene emissions in aphid infested tomatoes, increasing aphid parasitoid attraction (Coppola et al. 2017 ). Similarly, T. harzianum and T. atroviride improved maize tolerance to herbivores by modifying volatile terpene emissions that attract predators (Contreras-Cornejo et al. 2018 , 2021 ). The fungus T. atroviride also influenced S . littoralis and M. euphorbiae survival and growth rates via transcriptional changes in defense genes while enhancing parasitoid attraction through altered VOC profiles in tomato, even in the absence of herbivory (Coppola et al. 2019a ). Additionally, Fusarium solani strain K reduced Tetranychus urticae performance in tomato by modifying gene expression and VOC emissions, enhancing M. pygmaeus attraction (Pappas et al. 2018 ). Similarly, T. longibrachiatum increased M. pygmaeus attraction through HIPV modulation (Battaglia et al. 2013 ). However, in our study, T. virens colonization did not significantly alter maize VOC profiles, suggesting that the Trichoderma effects on tri-trophic interactions may be strain- and host-specific (Rodriguez & Redman 2008 ). In Y-tube olfactometer assays, M. pygmaeus exhibited a significant preference for maize HIPVs induced by H. armigera feeding, but fungal colonization did not further influence predator attraction to maize colonized by the fungal strains. This suggests that T. virens does not markedly alter VOC profiles in a way that affects third-trophic interactions. Similar findings were reported for T. harzianum , which also did not significantly modify tomato HIPVs or influence M. pygmaeus attraction (Meesters et al. 2024 ). Equally, fungal-derived volatiles did not affect the indirect defense of Brassica rapa against Pieris brassicae when plant roots were directly exposed to fungal VOCs, nor did they influence parasitism by Cotesia glomerata or alter the amount or quality of plant-emitted volatiles, resulting in no effects on indirect defense (Moisan et al. 2020 ). In conclusion, this study demonstrates that T. virens modulates plant-herbivore interactions but does not significantly affect plant-herbivore-predator dynamics. Our findings highlight the crucial role of fungal sesquiterpenes in enhancing maize resistance against H. armigera through direct defense mechanisms. To our knowledge, this is the first study to show that T. virens colonization negatively affects H. armigera performance via defense responses mediated by the vir4 gene cluster. These results underscore the potential of T. virens in strengthening maize resistance to herbivory and emphasize the need for further research into the genetic and metabolic pathways underlying T. virens -induced direct defense against H. armigera . Declarations Acknowledgments We gratefully acknowledge the financial support provided by the Deutsche Akademische Austauschdienst (DAAD) to the first author through the "Research Grants – Doctoral Programmes in Germany" program. Our sincere thanks go to Jonas Watterott for his assistance with GC-MS analyses. We also thank Christian Ulrich Baden from Bayer AG for providing H. armigera eggs used in this study. Author contribution N.N.: Conceptualization, experimental design, experiment execution, data analysis and visualization, methodology, and writing the original manuscript. M.R.: Project conception and conceptualization, validation of analyses, methodology, project administration, resource acquisition, supervision, manuscript review, writing, and editing. A.M.: Provided resources and supervision, reviewed the manuscript, and contributed revisions and comments. Funding Deutsche Akademische Austauschdienst (DAAD) provided a doctoral studies scholarship to the first author through the "Research Grants – Doctoral Programmes in Germany" program. All authors have agreed upon the final version of the manuscript. Competing interests The authors declare that they have no conflict of interest. References Alfano G, Ivey MLL, Cakir C, et al (2007) Systemic Modulation of Gene Expression in Tomato by Trichoderma hamatum 382. Phytopathology® 97:429–437. https://doi.org/10.1094/PHYTO-97-4-0429 Alınç T, Cusumano A, Peri E, et al (2021) Trichoderma harzianum Strain T22 Modulates Direct Defense of Tomato Plants in Response to Nezara viridula Feeding Activity. J Chem Ecol 47:455–462. https://doi.org/10.1007/s10886-021-01260-3 Alınç, T., Peri, E., Torta, L., Guarino, S., Colazza, S., Lievens, B., & Cusumano, A. (2024). Root inoculation with beneficial soil microbes enhances indirect plant defences induced by insect feeding and egg deposition. Functional Ecology. https://doi.org/10.1111/1365-2435.14594 Aartsma Y, Bianchi FJJA, Van Der Werf W, et al (2017) Herbivore‐induced plant volatiles and tritrophic interactions across spatial scales. New Phytol 216:1054–1063. https://doi.org/10.1111/nph.14475 Battaglia D, Bossi S, Cascone P, et al (2013) Tomato Below Ground–Above Ground Interactions: Trichoderma longibrachiatum Affects the Performance of Macrosiphum euphorbiae and Its Natural Antagonists. Mol Plant Microbe Interact 26:1249–1256. https://doi.org/10.1094/MPMI-02-13-0059-R. Brotman Y, Landau U, Cuadros-Inostroza Á, et al (2013) Trichoderma -Plant Root Colonization: Escaping Early Plant Defense Responses and Activation of the Antioxidant Machinery for Saline Stress Tolerance. PLoS Pathog 9:e1003221. https://doi.org/10.1371/journal.ppat.1003221 Contreras-Cornejo HA, Macías-Rodríguez L, del-Val E, Larsen J (2018) The root endophytic fungus Trichoderma atroviride induces foliar herbivory resistance in maize plants. Appl Soil Ecol 124:45–53. https://doi.org/10.1016/j.apsoil.2017.10.004 Contreras-Cornejo HA, Macías-Rodríguez L, del-Val E, Larsen J (2016) Ecological functions of Trichoderma spp. and their secondary metabolites in the rhizosphere: interactions with plants. FEMS Microbiol Ecol 92:fiw036. https://doi.org/10.1093/femsec/fiw036 Contreras-Cornejo HA, Macías-Rodríguez L, Real-Santillán RO, et al (2021) In a belowground multitrophic interaction, Trichoderma harzianum induces maize root herbivore tolerance against Phyllophaga vetula . Pest Manag Sci 77:3952–3963. https://doi.org/10.1002/ps.6415 Coppola M, Cascone P, Chiusano ML, et al (2017) Trichoderma harzianum enhances tomato indirect defense against aphids. Insect Sci 24:1025–1033. https://doi.org/10.1111/1744-7917.12475 Coppola M, Cascone P, Lelio ID, et al (2019a) Trichoderma atroviride P1 Colonization of Tomato Plants Enhances Both Direct and Indirect Defense Barriers Against Insects. Front Physiol 10. https://doi.org/doi: 10.3389/fphys.2019.00813 Coppola M, Diretto G, Digilio MC, et al (2019b) Transcriptome and Metabolome Reprogramming in Tomato Plants by Trichoderma harzianum strain T22 Primes and Enhances Defense Responses Against Aphids. Front Physiol 10: https://doi.org/10.3389/fphys.2019.00745 Dicke M, van Loon JJA, Soler R (2009) Chemical complexity of volatiles from plants induced by multiple attack. Nat Chem Biol 5:317–324. https://doi.org/10.1038/nchembio.169 Ebrahimi M, Mahdian K, De Clercq P (2019) Life‐history parameters and predation capacity of Macrolophus pygmaeus and Nesidiocoris tenuis (Hemiptera: Miridae) on eggs of Plutella xylostella (Lepidoptera: Plutellidae). Agric For Entomol 21:50–57. https://doi.org/10.1111/afe.12302 Fernandez-Conradi P, Jactel H, Robin C, et al (2018) Fungi reduce preference and performance of insect herbivores on challenged plants. Ecology 99:300–311. https://doi.org/10.1002/ecy.2044 Harman GE (2005) Overview of Mechanisms and Uses of Trichoderma spp. Phytopathology® 96:190–194. https://doi.org/10.1094/PHYTO-96-0190 Harman GE, Howell CR, Viterbo A, et al (2004) Trichoderma species — opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56. https://doi.org/10.1038/nrmicro797 Hermosa R, Viterbo A, Chet I, Monte E (2012) Plant-beneficial effects of Trichoderma and of its genes. Microbiology 158:17–25. https://doi.org/10.1099/mic.0.052274-0 Hermosa, R., Cardoza, R. E., Rubio, M. B., Gutiérrez, S., & Monte, E. (2014). Secondary Metabolism and Antimicrobial Metabolites of Trichoderma . In Biotechnology and Biology of Trichoderma (pp. 125–137). Elsevier. https://doi.org/10.1016/B978-0-444-59576-8.00010-2 Hilker M, Meiners T (2006) Early Herbivore Alert: Insect Eggs Induce Plant Defense. J Chem Ecol 32:1379–1397. https://doi.org/10.1007/s10886-006-9057-4 Holopainen JK, Gershenzon J (2010) Multiple stress factors and the emission of plant VOCs. Trends Plant Sci 15:176–184. https://doi.org/10.1016/j.tplants.2010.01.006 Ingegno BL, La-Spina M, Jordan MJ, et al (2016) Host Plant Perception and Selection in the Sibling Species Macrolophus melanotoma and Macrolophus pygmaeus (Hemiptera: Miridae). J Insect Behav 29:117–142. https://doi.org/10.1007/s10905-016-9549-1 Jiménez-Bremont, J. F., González-Pérez, E., Ortega-Amaro, M. A., Madrigal-Ortiz, S., Duque-Ortiz, A., & Mendoza-Mendoza, A. (2024). Volatile organic compounds emitted by Trichoderma : Small molecules with biotechnological potential. Scientia Horticulturae, 325, 112656. https://doi.org/10.1016/j.scienta.2023.112656 Kim J, Felton GW (2013) Priming of antiherbivore defensive responses in plants. Insect Sci 20:273–285. https://doi.org/10.1111/j.1744-7917.2012.01584.x Kramer R, Abraham W-R (2012) Volatile sesquiterpenes from fungi: what are they good for? Phytochem Rev 11:15–37. https://doi.org/10.1007/s11101-011-9216-2 Lelio ID, Coppola M, Comite E, et al (2021) Temperature Differentially Influences the Capacity of Trichoderma Species to Induce Plant Defense Responses in Tomato Against Insect Pests. Front Plant Sci 12:. https://doi.org/doi: 10.3389/fpls.2021.678830 Lelio ID, Forni G, Magoga G, et al (2023) A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota. 120: https://doi.org/10.1073/pnas.2216922120 Lins JC, van Loon JJA, Bueno VHP, et al (2014) Response of the zoophytophagous predators Macrolophus pygmaeus and Nesidiocoris tenuis to volatiles of uninfested plants and to plants infested by prey or conspecifics. BioControl 59:707–718. https://doi.org/10.1007/s10526-014-9602-y Macías-Rodríguez L, Contreras-Cornejo HA, Adame-Garnica SG, et al (2020) The interactions of Trichoderma at multiple trophic levels: inter-kingdom communication. Microbiol Res 240:126552. https://doi.org/10.1016/j.micres.2020.126552 Marra R, Ambrosino P, Carbone V, et al (2006) Study of the three-way interaction between Trichoderma atroviride , plant and fungal pathogens by using a proteomic approach. Curr Genet 50:307–321. https://doi.org/10.1007/s00294-006-0091-0 Maselou DA, Eirini Anastasaki, Panagiotis G. Milonas (2019) The Role of Host Plants, Alternative Food Resources and Herbivore Induced Volatiles in Choice Behavior of an Omnivorous Predator. Front Ecol Evol 6:. https://doi.org/10.3389/fevo.2018.00241 Massalha H, Korenblum E, Tholl D, Aharoni A (2017) Small molecules below-ground: the role of specialized metabolites in the rhizosphere. Plant J 90:788–807. https://doi.org/10.1111/tpj.13543 Meesters C, Weldegergis BT, Dicke M, et al (2024) Limited effects of plant-beneficial fungi on plant volatile composition and host-choice behavior of Nesidiocoris tenuis . Front Plant Sci. https://doi.org/10.3389/fpls.2023.1322719 Moisan K, Lucas‐Barbosa D, Villela A, et al (2020) No evidence of modulation of indirect plant resistance of Brassica rapa plants by volatiles from soil‐borne fungi. Ecol Entomol 45:1200–1211. https://doi.org/10.1111/een.12906 Monte E (2023). The sophisticated evolution of Trichoderma to control insect pests. Proceedings of the National Academy of Sciences, 120(12), e2301971120. https://doi.org/10.1073/pnas.2301971120 Morán-Diez ME, Martínez De Alba ÁE, Rubio MB, et al (2021) Trichoderma and the Plant Heritable Priming Responses. J Fungi 7:318. https://doi.org/10.3390/jof7040318 Nawrocka J (2013) Diversity in plant systemic resistance induced by Trichoderma . Biol Control 67:149–156. http://dx.doi.org/10.1016/j.biocontrol.2013.07.005 Nogueira-Lopez G, Greenwood DR, Middleditch M, et al (2018) The Apoplastic Secretome of Trichoderma virens During Interaction With Maize Roots Shows an Inhibition of Plant Defense and Scavenging Oxidative Stress Secreted Proteins. Front Plant Sci 9:409. https://doi.org/10.3389/fpls.2018.00409 Pangesti N, Pineda A, Pieterse CMJ, Dicke M (2013) Two-way plant-mediated interactions between root-associated microbes and insects: from ecology to mechanisms. Front Plant Sci 4. https://doi.org/0.3389/fpls.2013.00414 Pappas ML, Liapoura M, Papantoniou D, et al (2018) The Beneficial Endophytic Fungus Fusarium solani Strain K Alters Tomato Responses Against Spider Mites to the Benefit of the Plant. Front Plant Sci 9:1603. https://doi.org/10.3389/fpls.2018.01603 Pieterse CMJ, Zamioudis C, Berendsen RL, et al (2014) Induced Systemic Resistance by Beneficial Microbes. Annu Rev Phytopathol 52:347–375. https://doi.org/10.1146/annurev-phyto-082712-102340 Pineda A, Zheng S-J, van Loon JJA, et al (2010) Helping plants to deal with insects: the role of beneficial soil-borne microbes. Trends Plant Sci 15:507–514. https://doi.org/10.1016/j.tplants.2010.05.007 Poveda J (2021) Trichoderma as biocontrol agent against pests: New uses for a mycoparasite. Biol Control 159:104634. https://doi.org/10.1016/j.biocontrol.2021.104634 Rahman S, Michael Rostás, Ilka Vosteen (2024) Drought aggravates plant stress by favouring aphids and weakening indirect defense in a sugar beet tritrophic system. J Pest Sci 1–16. https://doi.org/10.1007/s10340-024-01799-6 Reino, J. L., Guerrero, R. F., Hernández-Galán, R., & Collado, I. G. (2007). Secondary metabolites from species of the biocontrol agent Trichoderma . Phytochemistry Reviews, 7(1), 89–123. https://doi.org/10.1007/s11101-006-9032-2 Rodriguez R, Redman R (2008) More than 400 million years of evolution and some plants still can’t make it on their own: plant stress tolerance via fungal symbiosis. J Exp Bot 59:1109–1114. https://doi.org/10.1093/jxb/erm342 Salwan, R., Rialch, N., & Sharma, V. (2019). Bioactive Volatile Metabolites of Trichoderma : An overview. In H. B. Singh, C. Keswani, M. S. Reddy, E. Sansinenea, & C. García-Estrada (Eds.), Secondary Metabolites of Plant Growth Promoting Rhizomicroorganisms (pp. 87–111). Springer Singapore. https://doi.org/10.1007/978-981-13-5862-3_5 Schausberger P, Peneder S, Jurschik S, Hoffmann D (2012) Mycorrhiza changes plant volatiles to attract spider mite enemies. Funct Ecol 26:441–449. https://doi.org/doi: 10.1111/j.1365-2435.2011. 01947.x Schweiger R, Padilla-Arizmendi F, Nogueira-López G, et al (2021) Insights into Metabolic Changes Caused by the Trichoderma virens –Maize Root Interaction. Mol Plant-Microbe Interactions® 34:524–537. https://doi.org/10.1094/MPMI-04-20-0081-R Shoresh M, Harman GE (2008) The Molecular Basis of Shoot Responses of Maize Seedlings to Trichoderma harzianum T22 Inoculation of the Root: A Proteomic Approach. Plant Physiol 147:2147–2163. https://doi.org/10.1104/pp.108.123810 Shoresh M, Harman GE, Mastouri F (2010) Induced Systemic Resistance and Plant Responses to Fungal Biocontrol Agents. Annu Rev Phytopathol 48:21–43. https://doi.org/10.1146/annurev-phyto-073009-114450 Silva DB, Urbaneja A, Pérez-Hedo M (2021) Response of mirid predators to synthetic herbivore-induced plant volatiles. Entomol Exp Appl 169:125–132. https://doi.org/10.1111/eea.12970 Thompson MN, Arriaga J, Bradford BJ, et al (2023) Belowground insect herbivory induces systemic volatile emissions that strengthen neighbouring plant resistance aboveground. Plant Cell Environ 47. https://doi.org/DOI: 10.1111/pce.14762 Turlings TCJ, Davison AC, TamÒ C (2004) A six-arm olfactometer permitting simultaneous observation of insect attraction and odor trapping. Physiol Entomol 29:45–55. https://doi.org/10.1111/j.1365-3032.2004.0362.x Turlings TC, Ton J (2006) Exploiting scents of distress: the prospect of manipulating herbivore-induced plant odours to enhance the control of agricultural pests. Curr Opin Plant Biol 9:421–427. https://doi.org/10.1016/j.pbi.2006.05.010 Urbaneja A, Montón H, Mollá O (2009) Suitability of the tomato borer Tuta absoluta as prey for Macrolophus pygmaeus and Nesidiocoris tenuis . J Appl Entomol 133:292–296. https://doi.org/10.1111/j.1439-0418.2008.01319.x Vinale, F., & Sivasithamparam, K. (2020). Beneficial effects of Trichoderma secondary metabolites on crops. Phytotherapy Research, 34(11), 2835–2842. https://doi.org/10.1002/ptr.6728 Vinale F, Sivasithamparam K, Ghisalberti EL, et al (2008) Trichoderma –plant–pathogen interactions. Soil Biol Biochem 40:1–10. https://doi.org/10.1016/j.soilbio.2007.07.002 Vinci G, Cozzolino V, Mazzei P, et al (2018) An alternative to mineral phosphorus fertilizers: The combined effects of Trichoderma harzianum and compost on Zea mays , as revealed by 1H NMR and GC-MS metabolomics. PLOS ONE 13:e0209664. https://doi.org/10.1371/journal.pone.0209664 Woo SL, Hermosa R, Lorito M, Monte E (2022) Trichoderma : a multipurpose, plant-beneficial microorganism for eco-sustainable agriculture. Nat Rev Microbiol 1–15. https://doi.org/10.1038/s41579-022-00819-5 Zeilinger, S., Gruber, S., Bansal, R., & Mukherjee, P. K. (2016). Secondary metabolism in Trichoderma – Chemistry meets genomics. Fungal Biology Reviews, 30(2), 74–90. https://doi.org/10.1016/j.fbr.2016.05.001 Zhou D, Huang X-F, Guo J, et al (2018) Trichoderma gamsii affected herbivore feeding behaviour on Arabidopsis thaliana by modifying the leaf metabolome and phytohormones. Microb Biotechnol 11:1195–1206. https://doi.org/10.1111/1751-7915.13310 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 13 Jan, 2026 Read the published version in Journal of Chemical Ecology → Version 1 posted Editorial decision: Revision requested 31 Oct, 2025 Reviews received at journal 19 Sep, 2025 Reviewers agreed at journal 21 Aug, 2025 Reviews received at journal 19 Aug, 2025 Reviewers agreed at journal 02 May, 2025 Reviewers invited by journal 02 May, 2025 Editor assigned by journal 01 May, 2025 Submission checks completed at journal 29 Apr, 2025 First submitted to journal 28 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6548157","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451180499,"identity":"26f6fa60-463b-4ed4-83e3-7871275299ac","order_by":0,"name":"Noor Agha Nawakht","email":"","orcid":"","institution":"University of Göttingen","correspondingAuthor":false,"prefix":"","firstName":"Noor","middleName":"Agha","lastName":"Nawakht","suffix":""},{"id":451180500,"identity":"84d1b524-42d4-45c8-bc9d-02fd3e038e5a","order_by":1,"name":"Artemio Mendoza-Mendoza","email":"","orcid":"","institution":"Lincoln University","correspondingAuthor":false,"prefix":"","firstName":"Artemio","middleName":"","lastName":"Mendoza-Mendoza","suffix":""},{"id":451180501,"identity":"4ffd8a93-48d1-4eed-b397-fde7f313d368","order_by":2,"name":"Michael Rostás","email":"data:image/png;base64,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","orcid":"","institution":"University of Göttingen","correspondingAuthor":true,"prefix":"","firstName":"Michael","middleName":"","lastName":"Rostás","suffix":""}],"badges":[],"createdAt":"2025-04-28 13:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6548157/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6548157/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10886-025-01681-4","type":"published","date":"2026-01-13T16:30:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82180814,"identity":"15553bda-741f-4dde-bf9a-473317a88259","added_by":"auto","created_at":"2025-05-07 11:52:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117337,"visible":true,"origin":"","legend":"\u003cp\u003ePerformance of \u003cem\u003eH. armigera\u003c/em\u003e on maize seedlings colonized by fungal strains. (\u003cstrong\u003eA\u003c/strong\u003e) Caterpillar weight gain. The Y-axis represents caterpillar weight, while the X-axis shows days after feeding. Data were analyzed using a linear mixed-effects model, followed by Tukey's HSD test (p ≤ 0.05) for mean separation. Different letters above the lines indicate significant differences among treatments. (\u003cstrong\u003eB\u003c/strong\u003e) Survival rate of \u003cem\u003eH. armigera\u003c/em\u003e caterpillars over the 9-day experimental period. Kaplan-Meier survival curves with log-rank test (p ≤ 0.05) showed no significant differences among treatments (p \u0026gt; 0.05). Data for surviving caterpillars were censored after day 9. n.s.= not significant. Treatments: MH = non-colonized maize infested with \u003cem\u003eH. armigera\u003c/em\u003e caterpillars (control), MVH = maize inoculated with the vir4 mutant and infested with caterpillars, MWH = maize inoculated with wild-type \u003cem\u003eT. virens\u003c/em\u003e and infested with caterpillars (n = 24).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6548157/v1/c77ada6994e0333e79eba918.png"},{"id":82181403,"identity":"9478b9ca-4c10-4cf4-b8f3-cda193f1f098","added_by":"auto","created_at":"2025-05-07 12:00:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":677826,"visible":true,"origin":"","legend":"\u003cp\u003eVolatile organic compounds profile of maize seedlings subjected to different treatments. (\u003cstrong\u003eA\u003c/strong\u003e) Box-whisker plots displaying log-transformed data on total volatile emission for each treatment. Medians are shown as horizontal lines, interquartile ranges (IQR) are indicated by boxes, whiskers extend to 1.5 times the IQR, and outliers are represented by dots outside the whiskers. The mean for each treatment is marked by a yellow square with black outline. Different letters denote statistically significant differences between treatments as determined by ANOVA and Tukey’ at p ≤ 0. 05. (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap illustrating the abundance and clustering of VOCs across various treatments. The color intensity represents the relative concentration of each compound. Treatments include: M = uninfested control maize, MH = maize infested with \u003cem\u003eH. armigera\u003c/em\u003ecaterpillars, MVH = maize inoculated with mutant and infested with caterpillars, and MWH = maize inoculated with wild-type and infested with caterpillars. (\u003cstrong\u003eC\u003c/strong\u003e) Principal Component Analysis score plot showing the variance explained by the first two principal components (PC1 and PC2), which account for 45.29% and 27.16% of the total variance, respectively\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6548157/v1/1e2dfce9e542857d249c2a82.png"},{"id":82180818,"identity":"49b7e08d-91d1-4541-a3ac-8351bcbfd5a1","added_by":"auto","created_at":"2025-05-07 11:52:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":170184,"visible":true,"origin":"","legend":"\u003cp\u003eResponse of \u003cem\u003eM. pygmaeus\u003c/em\u003e to maize volatiles in a Y-tube olfactometer. The bars represent the percentage of responding insects (n = 30) for different treatment combinations: \u003cstrong\u003eM\u003c/strong\u003e= uninfested control maize, \u003cstrong\u003eMH\u003c/strong\u003e = maize infested with \u003cem\u003eH. armigera\u003c/em\u003e caterpillars, \u003cstrong\u003eMV\u003c/strong\u003e = maize inoculated with mutant, \u003cstrong\u003eMVH\u003c/strong\u003e = mutant-inoculated maize infested with caterpillars, \u003cstrong\u003eMW\u003c/strong\u003e= maize inoculated with wild type, and \u003cstrong\u003eMWH\u003c/strong\u003e = wild-type-inoculated maize infested with caterpillars. Predator response rates are shown as percentages on the right side of the plot. Pairwise comparisons between treatments were performed using Chi-squared (χ²) tests to assess predator preference, with significant differences (p ≤ 0.05) indicated by asterisks (*).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6548157/v1/f8fcbf42a5808ef3467a61c0.png"},{"id":100614869,"identity":"42fd8f10-29aa-4b1f-ae35-833852303ee6","added_by":"auto","created_at":"2026-01-19 17:27:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1730442,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6548157/v1/721a5864-471e-409d-9bc8-46a0407f1a52.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSesquiterpene Biosynthetic Gene \u003cem\u003evir4 \u003c/em\u003efrom \u003cem\u003eTrichoderma virens \u003c/em\u003eEnhances Direct Herbivore Resistance while Maintaining Indirect Defense\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe genus \u003cem\u003eTrichoderma\u003c/em\u003e (teleomorph Hypocrea) is a globally distributed soil-borne fungus that thrives in diverse habitats (Woo et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It plays a key role in sustainable agriculture, acting as a biopesticide, bio-stimulant, and plant growth promoter while enhancing plant defenses (Harman et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Woo et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As an opportunistic symbiont, \u003cem\u003eTrichoderma\u003c/em\u003e colonizes the rhizosphere and the apoplast of plant roots, inhabiting intercellular spaces and outer cell layers (Harman et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Hermosa et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Brotman et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nogueira-Lopez et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Traditionally, its biocontrol activity was attributed to mycoparasitism, antibiosis, and competition for space and resources (Harman \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Vinale et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, recent studies highlight its role in inducing systemic and localized plant defense responses (Nawrocka \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Poveda \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Monte \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eTrichoderma\u003c/em\u003e spp. produce a diverse array of volatile and non-volatile secondary metabolites that contribute to their bioactivity and defense-inducing properties in host plants (Hermosa et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Jim\u0026eacute;nez-Bremont et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Reino et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Salwan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vinale \u0026amp; Sivasithamparam. 2020; Zeilinger et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eColonization of maize roots by \u003cem\u003eTrichoderma\u003c/em\u003e spp. can significantly alter the host plant\u0026rsquo;s metabolome (Vinale et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Vinci et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), proteome, and transcriptome (Harman \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Marra et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Alfano et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Shoresh \u0026amp; Harman \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Nogueira-Lopez et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Schweiger et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) demonstrated that the wild-type \u003cem\u003eT. virens\u003c/em\u003e strain Gv29.8 and its \u003cem\u003eΔvir4\u003c/em\u003e mutant, which is deficient in sesquiterpene biosynthesis, differentially modulate maize root gene expression and metabolic composition. Their study revealed that \u003cem\u003eT. virens\u003c/em\u003e colonization profoundly affects several metabolites derived from the shikimate pathway, including flavonoids and other compounds, in a genotype-dependent manner. These metabolic changes may influence not only belowground plant-insect interactions but also aboveground interactions, as \u003cem\u003eTrichoderma\u003c/em\u003e spp. can induce systemic resistance in leaves, potentially triggering cascading effects across trophic levels. Several studies have reported \u003cem\u003eTrichoderma\u003c/em\u003e-induced resistance against herbivorous insects from different feeding guilds, including lepidopterans (Contreras-Cornejo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Coppola et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Mac\u0026iacute;as-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, \u003cem\u003eT. atroviride\u003c/em\u003e reduced aphid and caterpillar fitness in maize and tomato by modulating plant defense mechanisms (Coppola et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). Similarly, \u003cem\u003eT. harzianum\u003c/em\u003e negatively affected \u003cem\u003eNezara viridula\u003c/em\u003e feeding behavior in tomato by enhancing direct defense responses (Alinc et al. 2021).\u003c/p\u003e \u003cp\u003ePlants communicate with their environment through volatile organic compounds (VOCs), which can mediate both above- and belowground interactions (Massalha et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Thompson et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Insect feeding, in particular, triggers the release of herbivore-induced plant volatiles (HIPVs), which play a critical role in plant defense by attracting natural enemies of herbivores (Aartsma et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hilker \u0026amp; Meiners. 2006; Dicke et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Microbial colonization, including beneficial fungi, can influence the emission of HIPVs by modifying both the composition and quantity of emitted volatiles. These changes, in turn, may affect the behavior and effectiveness of natural enemies in locating their herbivorous prey (Holopainen \u0026amp; Gershenzon. 2010; Pangesti et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pineda et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Fernandez-Conradi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Schausberger et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For instance, \u003cem\u003eTrichoderma harzianum\u003c/em\u003e T22 was shown to enhance VOC-mediated defenses by increasing the attraction of aphid parasitoids in tomato (Coppola et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, \u003cem\u003eT. harzianum\u003c/em\u003e and \u003cem\u003eT. atroviride\u003c/em\u003e strain IMI 206040 improve maize tolerance to herbivores by modifying the emission of volatile terpenes that attract predators (Contreras-Cornejo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, \u003cem\u003eT. harzianum\u003c/em\u003e primes tomato plants to emit VOCs that attract parasitoids of aphids and stink bugs (Coppola et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Alinc et al. 2024).\u003c/p\u003e \u003cp\u003e \u003cem\u003eH. armigera\u003c/em\u003e (Lepidoptera: Noctuidae) is a polyphagous caterpillar that infests economically important crops, including maize (\u003cem\u003eZea mays\u003c/em\u003e L.). Its high fecundity, broad host range, and facultative diapause contribute to its status as a major pest. The zoophytophagous mirid bug \u003cem\u003eM. pygmaeus\u003c/em\u003e (Hemiptera: Miridae) is an efficient predator of various herbivorous pest insect including \u003cem\u003eH. armigera\u003c/em\u003e and is attracted to HIPVs emitted by crops such as tomato and tobacco (Maselou et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ingegno et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Urbaneja et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ebrahimi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, no studies have examined \u003cem\u003eM. pygmaeus\u003c/em\u003e attraction to maize HIPVs particularly in the presence of \u003cem\u003eT. virens\u003c/em\u003e strains as endophytic root colonizers.\u003c/p\u003e \u003cp\u003eSince \u003cem\u003eT. virens\u003c/em\u003e and its \u003cem\u003evir4\u003c/em\u003e knockout mutant influence maize root metabolic profile (Schweiger et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), we hypothesized that these changes could extend systemically to higher trophic levels, affecting herbivores and their natural enemies aboveground. Therefore, this study aimed to investigate: (i) how maize root colonization by both \u003cem\u003eT. virens\u003c/em\u003e genotypes affects \u003cem\u003eH. armigera\u003c/em\u003e survival and weight gain, (ii) whether fungal colonization alters maize VOC composition or quantity and (iii) the consequences of these changes for \u003cem\u003eM. pygmaeus\u003c/em\u003e foraging behavior within this multitrophic system.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003eInsects rearing.\u003c/b\u003e Eggs of \u003cem\u003eH. armigera\u003c/em\u003e were supplied by Bayer AG Crop Sciences (Monheim, Germany), and the larvae were reared on an artificial diet. Eggs were placed in plastic containers with slices of the diet and kept in an environmental chamber at 23\u0026deg;C, 65\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, and a 14-hour light/10-hour dark photoperiod to induce hatching. After hatching, the neonates were reared on artificial diet until they reached the desired developmental stage, after which they were used in experiments. Adults of \u003cem\u003eM. pygmaeus\u003c/em\u003e were obtained from Katz Biotech AG (Germany), along with \u003cem\u003eSitotroga\u003c/em\u003e eggs as supplementary food. A colony was established in BugDorm insect-rearing cages (BugDorm\u0026reg;, MegaView Science Co., Ltd.Taiwan). Potato tubers were placed on moistened soil in a tray inside the cages, and \u003cem\u003eSitotroga\u003c/em\u003e eggs were provided in a small Petri dish. Male and female adults of \u003cem\u003eM. pygmaeus\u003c/em\u003e were released into the cage for mating and reproduction. Emerging adults were continuously transferred to new cages, maintained at ambient room temperature.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFungal culture and inoculum preparation\u003c/b\u003e. The fungal strains were cultured, and inoculum was prepared as described by Schweiger et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Two genotypes of the \u003cem\u003eT. virens\u003c/em\u003e strain Gv29.8 were used in the study: the wild-type and the \u003cem\u003eΔvir4\u003c/em\u003e knockout mutant, which is defective in sesquiterpene biosynthesis, hereinafter referred to as \u0026ldquo;wild-type\u0026rdquo; and \u0026ldquo;mutant\u0026rdquo;, respectively. Both fungal strains were propagated on potato dextrose agar (PDA) (Carl Roth GmbH, Karlsruhe, Germany). Regular sub-culturing was done in sterile Petri dishes containing PDA, and the plates were kept in an environmental chamber under controlled conditions of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 65% relative humidity, and a 12-hour light: 12-hour dark cycle for 7 days to promote conidiation. To harvest the conidia, fungal plates were flooded with 15\u0026ndash;20 ml of sterile water using a pipette. A microscope slide was used to gently scrape the surface of the media plate to detach the conidia from the mycelium. Using a funnel, the resulting suspension was then filtered through a cheesecloth into an Erlenmeyer flask. The final concentration of conidia for each fungal strain was adjusted to 1x10\u003csup\u003e^6\u003c/sup\u003e conidia/ml to be used in subsequent inoculation procedures.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlant material and fungal inoculation.\u003c/b\u003e Seeds of maize, hybrid line 34H31 (Pioneer\u0026reg; Brand Products, Gisborne, New Zealand), were used in the experiments, and the inoculation procedure followed Nogueira-Lopez et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Prior to inoculation, the seeds were surface sterilized by submerging them in 2% sodium hypochlorite (NaOCl) solution, followed by 70% ethanol for 7 min each. The sterilization process was followed by five washes with autoclaved tap water. The success of sterilization was confirmed by plating 100 \u0026micro;L of water from the final rinse on PDA (Carl Roth GmbH, Karlsruhe, Germany) and incubating under the same conditions used for fungal culture preparation. Seeds were placed in Petri dishes with moistened sterilized-filter paper to pre-germinate overnight. The following day, seeds were inoculated with the fungal spore suspension individually at a conencetration of 1x10^\u003csup\u003e6\u003c/sup\u003e conidia under clean bench. Three treatment groups were prepared: (1) seeds inoculated with \u003cem\u003eT. virens\u003c/em\u003e wild-type strain, (2) seeds inoculated with the mutant strain, and (3) a control group where kernels were mock-inoculated with sterilized water using the same method as the fungal spore treatment. All procedures were conducted under sterile conditions to prevent contamination. Treated seeds were sown in plastic pots containing gamma-irradiated soil. The pots were maintained in climate-controlled chambers with 80% relative humidity, a 16:8 light-dark cycle, and a temperature of 25\u0026deg;C to allow for proper growth of the maize plants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHerbivore performance bioassay\u003c/b\u003e. To assess the impact of fungal colonization on herbivore survival and weight gain, 2nd instar larvae of \u003cem\u003eH. armigera\u003c/em\u003e were weighed and randomly assigned to 7-days old maize seedlings. Each seedling received a single larva placed into the whorl. The total sample size was 24 seedlings per treatment group (control, wild-type, mutant). To prevent larval escape and ensure airflow, each potted seedling was covered with a perforated cellophane plastic bag. The plants were then placed in an environmental chamber with 25\u0026deg;C, a 16-hour light/8-hour dark photoperiod, and 80% relative humidity. The caterpillars were weighed again 3, 6, and 9 days post infestation (dpi)\u0026mdash;to determine the weight gain across different treatments. Additionally, survival was monitored daily for each treatment group throughout the 9-day feeding period to track mortality rates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollection and analyses of the volatile blend\u003c/b\u003e. To collect and analyze the VOCs emitted by maize seedlings treated with \u003cem\u003eT. virens\u003c/em\u003e wild-type, mutant, or control, a push-pull dynamic headspace VOCs collection system was employed using the lower part of a six-arm olfactometer (Turlings et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Maize seedlings were infested with ten 2nd instar larvae of \u003cem\u003eH. armigera\u003c/em\u003e one day before volatile collection. The VOCs were collected using volatile trapping filters containing 30 mg of 80\u0026ndash;100 mesh Porapak Q (Volatile Collection Trap LLC, FL, USA), attached to the outlet air pipe of the odor source vessels. A central in-house compressor provided humidified and filtered air at an inflow rate of 1 l/min. Air was pulled out from the odor source vessels at a flow rate of 0.8 l/min, using a membrane pump. The VOCs collection started daily at 10:00 a.m. and lasted for four hours. All equipment, including glassware and vessels, was thoroughly cleaned with demineralized water, 99.5% acetone, and oven-dried at 180\u0026deg;C for two hours to prevent contamination. The filters were washed with 1 ml of dichloromethane (DCM) before use as described (Rahman et al. 2024). After VOCs collection, the trapped VOCs were eluted from the filters by rinsing them with 150 \u0026micro;l of DCM into 1 ml glass vials. The samples were stored at -80\u0026deg;C for further analysis, with 200 ng of 1,2,3,4-tetrahydronaphthalene added as an internal standard. For analysis, 40 \u0026micro;l of the final solution was transferred to glass vials with glass inserts for the GC-MS autosampler. A 2 \u0026micro;l aliquot was injected in pulsed splitless mode into the GC-MS system (5977B HES MSD, Agilent Technologies). The GC oven temperature was initially set at 40\u0026deg;C for 3 min, then increased gradually at a rate of 8\u0026deg;C min⁻\u0026sup1; to 320\u0026deg;C, which was held for 10 min. Helium was used as the carrier gas at a constant flux of 1.5 ml/min. Chromatograms were automatically integrated using Agilent MSD ChemStation software and mass spectra were compared to those in the libraries NIST17 and Wiley11 for tentative identification. For further confirmation of compound identities, the retention index (RI) of each compound was calculated and compared to the RI values listed in the Van Den Dool and Kratz RI Table available in the NIST Chemistry Webbook library. Quantification of the compounds was based on their mass peak areas relative to the peak area of the internal standard. This method allowed for relative comparisons of the amounts of each volatile emitted in the treatments. The experimental treatments consisted of the following setups: (1) maize, (2) maize infested with herbivores, (3) maize colonized by the wild-type and infested with herbivores, (4) maize colonized by the mutant and infested with herbivores, along with a blank (empty collection vessel to control for background contamination).\u003c/p\u003e \u003cp\u003e \u003cb\u003eResponse of M. pygmaeus to Herbivore-Induced Maize Volatiles.\u003c/b\u003e Y-tube olfactometer assays were conducted to assess the response of \u003cem\u003eM. pygmaeus\u003c/em\u003e to volatiles emitted by maize treated with fungi and/or herbivores. The Y-tube had an 8 mm inner diameter, a 20 cm length for both the entry and side arms, and a 70\u0026deg; angle between the side arms. The methods were adapted from Lins et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), Maselou et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and Silva et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Maize seedlings (treated with fungal strains or left untreated as control) were infested with ten 2nd instar \u003cem\u003eH. armigera\u003c/em\u003e larvae one day before the experiment to induce the release of VOCs. Seedlings were enclosed in plastic bags sealed at both ends. Two plastic tubes were inserted into the bags: one to allow inlet air from the air pump and the other to connect to the arms of the Y-tube olfactometer. Humidified and charcoal-filtered air was provided by an air compressor (Stimulus Controller CS-55 V2, Ockenfels Syntech GmbH, Germany), with the airflow adjusted to 0.3 L min⁻\u0026sup1;. Adults of \u003cem\u003eM. pygmaeus\u003c/em\u003e were starved for 18 h before the experiment. Each individual insect was introduced into the central arm of the olfactometer and observed for a maximum of 10 min to make a choice by moving into one of the arms connected to an odor source. Insects moving 10 cm past the branching point towards one of the odor sources were considered to have made a choice. Insects that did not select a side within the 10-min period were classified as unresponsive and were excluded from the analysis. Five adult female insects were tested per plant set, and after each test, the olfactometer and odor sources were replaced. The control treatment followed the same procedure but without herbivore infestation. Before use, all olfactometer components were cleaned with Contrad 70\u0026reg; detergent (Decon Laboratories Limited, VWR International, Hove, East Sussex, UK), rinsed with distilled water, and air-dried overnight at room temperature. Experiments were conducted at room temperature under a fume hood, and to avoid positional bias, the positions of the odor sources (infested vs. healthy) were alternated between the left and right side arms.\u003c/p\u003e \u003cp\u003eTo compare the preferences of \u003cem\u003eM. pygmaeus\u003c/em\u003e for different volatile profiles, six treatment combinations were tested pairwise using the Y-tube olfactometer. The treatment comparisons were as follows: (1) maize vs. maize infested with herbivores (M vs. MH); (2) maize vs. maize inoculated with the wild-type (M vs. MW); (3) maize vs. maize inoculated with the \u003cem\u003evir4\u003c/em\u003e mutant (M vs. MV); (4) maize infested with herbivores vs. maize inoculated with wild-type and infested with herbivores (MH vs. MWH); (5) maize infested with herbivores vs. maize inoculated with mutant and infested with herbivores (MH vs. MVH) (6) maize inoculated with wild-type and infested with herbivores vs. maize inoculated with mutant and infested with herbivores (MWH vs. MVH). Individuals of \u003cem\u003eM. pygmaeus\u003c/em\u003e were tested only once, with a total of 30 insects per treatment pair. The positions of the odor sources connected to the olfactometer were exchanged after testing five insects, and seedlings were discarded after each test.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData analyses and statistical procedures\u003c/b\u003e. All statistical analyses and data visualizations were performed using R. A linear mixed-effects model was fitted to assess the effects of treatment, time, and their interaction on caterpillar weight gain. Treatment and time were treated as fixed effects, while individual caterpillars were considered random effects to account for repeated measures. Tukey\u0026rsquo;s Honestly Significant Difference (HSD) test, adjusted for multiple comparisons, was used for post-hoc mean comparisons. These analyses were conducted using the \u003cem\u003elme4\u003c/em\u003e, \u003cem\u003ecar\u003c/em\u003e, and \u003cem\u003eemmeans\u003c/em\u003e packages in R. The relevant statistical assumptions were thoroughly evaluated. Normality of residuals was assessed through graphical inspections and the Shapiro-Wilk test, while homoscedasticity was evaluated using Levene's test. Kaplan-Meier survival analysis, along with the log-rank test, was used to determine statistical differences in caterpillar survival across treatments. For the analysis of volatile emissions, the total volatile emission data were log-transformed to meet the assumptions of ANOVA and Tukey\u0026rsquo;s HSD test was conducted for mean separation. The quantities of individual VOCs among treatments were compared using the Kruskal-Wallis non-parametric test followed by Dunn test with a Bonferroni correction. Heatmap was generated using the \u003cem\u003epheatmap\u003c/em\u003e package in R and color gradients were customized using the \u003cem\u003eRColorBrewer\u003c/em\u003e package. To test the null hypothesis of no preference by the predator between the two odor sources, a Chi-squared (χ\u0026sup2;) test was performed. A significance threshold of p\u0026thinsp;\u0026le;\u0026thinsp;0.05 was applied for all statistical tests.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eHerbivore performance and survival\u003c/b\u003e. Larval performance was evaluated for each treatment by monitoring weight gain at various time points post-feeding. Statistically significant differences in fresh body mass were observed among caterpillars fed on different treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A linear mixed-effects model revealed significant main effects for both treatment (F\u003csub\u003e2.64, 11\u003c/sub\u003e = 3.717, p\u0026thinsp;=\u0026thinsp;0.02) and day (F\u003csub\u003e3, 177.31\u003c/sub\u003e = 159.68, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as well as a significant interaction between treatment and day (F\u003csub\u003e6, 177.31\u003c/sub\u003e = 3.026, p\u0026thinsp;=\u0026thinsp;0.007). Further pairwise comparisons showed that by day 9, caterpillars feeding on maize seedlings colonized by the wild-type genotype had significantly lower body weights compared to those on both the control (p\u0026thinsp;=\u0026thinsp;0.01) and mutant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). On the other hand, no significant differences in caterpillar survival were observed among the treatments over the 9-days experimental period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (Log-Rank test, χ\u0026sup2; = 1.63, df\u0026thinsp;=\u0026thinsp;2, P\u0026thinsp;=\u0026thinsp;0.44). The experiments were terminated on day 9, as some seedlings in certain treatments (i.e., control or mutant) were fully consumed by the caterpillars towards the 9th day.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eVOCs emission.\u003c/b\u003e Herbivory induced a distinct volatile bouquet, with some qualitative differences attributed to the fungal strains. Comparing the wild-type and mutant treatments, certain compounds were detected in one treatment but absent in the other. For instance, (\u003cem\u003eZ\u003c/em\u003e)-3-hexen-1-yl acetate was not found in the wild-type treatment but was present in the mutant treatment. Additionally, α‑bergamotene was identified in both fungal treatments but was absent in the herbivore-only treatment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the quantity of individual VOCs did not differ statistically among treatments. ANOVA revealed significant differences in total volatile emissions between treatments (F\u003csub\u003e3, 24\u003c/sub\u003e = 3.18, p\u0026thinsp;=\u0026thinsp;0.04; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A Tukey post-hoc test indicated that healthy maize emitted significantly fewer volatiles compared to the mutant-inoculated treatment (p\u0026thinsp;=\u0026thinsp;0.03), while differences with other treatments were not statistically significant. Only trace amounts of VOCs were released from control maize. A heatmap visualization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) illustrates the variation in volatile profiles across treatments, suggesting that fungal colonization may have led to qualitative changes in the plant's volatile profile, albeit to a limited extent. Notably, herbivore-infested maize treated with the fungal mutant exhibited the highest VOC emissions, including δ-cadinene, (\u003cem\u003eE\u003c/em\u003e)-4, 8-dimethylnona-1, 3,7-triene (DMNT), and ylangene, Similarly, the wild-type treatment also showed elevated levels of certain VOCs, such as delta-cadinene, DMNT, and farnesene, although in lower quantities compared to the mutant treatment. Hierarchical clustering from the heatmap indicates that the treatments colonized by fungal strains and subjected to herbivory shared a somewhat similar volatile profile, as reflected by their clustering together. Furthermore, the principal component analysis (PCA) score plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) shows clear separation between the treatments. PC1 explains 45.29% of the total variance, while PC2 accounts for 27.16%, together explaining 72.45% of the total variance. Particularly, the overlap between the mutant-herbivory and wild-type-herbivory treatments suggests that both treatments induce similar, yet slightly distinct, VOC responses, indicating their potential role in modulating the maize VOCs profile. Note, that untreated maize was not included in the analysis as most compounds were absent.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEstimated total VOC emission rate (ng/h/plant) from maize seedlings under different treatments: M\u0026thinsp;=\u0026thinsp;uninfested control maize, MH\u0026thinsp;=\u0026thinsp;maize infested with \u003cem\u003eH. armigera\u003c/em\u003e caterpillars, MVH\u0026thinsp;=\u0026thinsp;maize inoculated with the mutant and infested with caterpillars, and MWH\u0026thinsp;=\u0026thinsp;maize inoculated with the wild-type and infested with caterpillars. Single VOCs were compared among treatments using the Kruskal-Wallis non-parametric test. Values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE), n\u0026thinsp;=\u0026thinsp;7. Value in the same row sharing the same letters are not statistically different at p\u0026thinsp;\u0026le;\u0026thinsp;0.05. n.d.= not detected.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eTotal VOCs emission ng/h/plant\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMVH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMWH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(Z)-3-Hexen-1-yl acetate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.97\u0026thinsp;\u0026plusmn;\u0026thinsp;5.97\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.71\u0026thinsp;\u0026plusmn;\u0026thinsp;5.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.12\u0026thinsp;\u0026plusmn;\u0026thinsp;6.19 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyclosativene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYlangene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-Bergamotene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-Farnesene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eδ-Cadinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOlfactometer response of the predator.\u003c/b\u003e The response of \u003cem\u003eM. pygmaeus\u003c/em\u003e to volatile blends released by maize seedlings colonized by different fungal strains, with or without herbivory, was assessed using a Y-tube olfactometer. The choices made by the predator, represented as percentages, indicate their preference for one arm of the olfactometer over the other. Predators significantly preferred the arm containing herbivore-damaged maize seedlings as the odor source compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When fungal inoculation was also involved, the predators exhibited a significant preference for herbivore-infested maize seedlings over uninfested controls, regardless of fungal inoculation (χ\u0026sup2; = 4.17, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.04) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, Chi-squared tests indicated no significant preference for volatile blends associated with different fungal treatments used in different combinations. Specifically, predator preference did not differ significantly between maize inoculated with the wild-type and infested with caterpillars (MWH) and maize inoculated with the mutant and infested with caterpillars (MVH) (χ\u0026sup2; = 1.09, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.29). Similarly, no significant differences were observed between uninfested control maize (M) and wild-type-inoculated maize (MW) (χ\u0026sup2; = 0.20, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.65); uninfested control maize (M) and mutant-inoculated maize (MV) (χ\u0026sup2; = 0.40, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.82); herbivore-infested maize (MH) and wild-type-inoculated, herbivore-infested maize (MWH) (χ\u0026sup2; = 0.36, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.54); or herbivore-infested maize (MH) and mutant-inoculated, herbivore-infested maize (MVH) (χ\u0026sup2; = 0.04, df\u0026thinsp;=\u0026thinsp;1, P\u0026thinsp;=\u0026thinsp;0.83).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cem\u003eTrichoderma\u003c/em\u003e spp. are widely recognized as effective biocontrol agents against phytopathogens, yet their role in mediating plant-insect interactions remains underexplored (Poveda \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lelio et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Building on the findings of Schweiger et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which demonstrated that colonization of maize roots by wild-type and mutant \u003cem\u003eT. virens\u003c/em\u003e differentially alters the plant root metabolome, this study investigated whether these metabolic changes influence the development and survival of \u003cem\u003eH. armigera\u003c/em\u003e caterpillars aboveground. Additionally, we assessed the effects of \u003cem\u003eT. virens\u003c/em\u003e colonization on indirect defense mechanisms involving the predator \u003cem\u003eM. pygmaeus\u003c/em\u003e. Our results demonstrate that maize colonized by wild-type \u003cem\u003eT. virens\u003c/em\u003e significantly inhibited \u003cem\u003eH. armigera\u003c/em\u003e caterpillar development compared to mutant-colonized and control plants, suggesting that \u003cem\u003eT. virens\u003c/em\u003e enhances direct systemic resistance in maize. This effect is likely mediated by fungal sesquiterpenes encoded by the \u003cem\u003evir4\u003c/em\u003e gene cluster, underscoring their importance in plant defense.\u003c/p\u003e \u003cp\u003eInduced systemic resistance is a key defense mechanism in plants, wherein beneficial rhizosphere microbes, such as \u003cem\u003eTrichoderma\u003c/em\u003e spp., prime plants for enhanced defense against pathogens and herbivores (Pieterse et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eTrichoderma\u003c/em\u003e spp. produce a diverse array of bioactive secondary metabolites, including sesquiterpenes, which play critical roles in plant-microbe and plant-insect interactions (Kramer \u0026amp; Abraham \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Vinale et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Reino et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These metabolites act as signaling molecules and priming agents, systemically enhancing plant defense responses when released into the rhizosphere (Contreras-Cornejo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shoresh et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pieterse et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The poor performance of \u003cem\u003eH. armigera\u003c/em\u003e on maize colonized by wild-type \u003cem\u003eT. virens\u003c/em\u003e may thus be attributed to the priming or induction of plant defenses by sesquiterpenes encoded by the \u003cem\u003evir4\u003c/em\u003e gene cluster. This aligns with previous reports of \u003cem\u003eTrichoderma\u003c/em\u003e-mediated host defense priming in various crops against herbivores across different feeding guilds (Alın\u0026ccedil; et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Coppola et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Mor\u0026aacute;n-Diez et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, also direct deterrence of the herbivore by \u003cem\u003evir4\u003c/em\u003e-derived metabolites that may be translocated from root to shoot cannot be ruled out and merits further investigation.\u003c/p\u003e \u003cp\u003eOur findings are align with previous research on \u003cem\u003eTrichoderma\u003c/em\u003e-plant-insect interactions. For instance, \u003cem\u003eT. gamsii\u003c/em\u003e altered the foliar metabolome of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, reducing weight gain in \u003cem\u003eTrichoplusia ni\u003c/em\u003e (Zhou et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, \u003cem\u003eT. atroviride\u003c/em\u003e colonization limited herbivory by \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e through the induction of plant resistance, which was partly mediated by fungal-derived antifeedant secondary metabolites (Contreras-Cornejo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It also negatively impacted the development of \u003cem\u003eS. littoralis\u003c/em\u003e and aphids via transcriptional changes in defense-related genes (Coppola et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). Inoculation with \u003cem\u003eT. harzianum\u003c/em\u003e reduced the growth rate of \u003cem\u003eNezara viridula\u003c/em\u003e by enhancing direct defense mechanisms and priming tomato plants (Alın\u0026ccedil; et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) while also enhancing maize root resistance to \u003cem\u003ePhyllophaga vetula\u003c/em\u003e through the antifeedant activity of fungal-derived 6-pentyl-2H-pyran-2-one (6-PP) (Contreras-Cornejo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, \u003cem\u003eT. afroharzianum\u003c/em\u003e and \u003cem\u003eT. atroviride\u003c/em\u003e have been shown to enhance tomato resistance against \u003cem\u003eS. littoralis\u003c/em\u003e and \u003cem\u003eM. euphorbiae\u003c/em\u003e (Lelio et al. 2022, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Collectively, these findings underscore the potential of \u003cem\u003eT. virens\u003c/em\u003e in enhancing maize direct systemic resistance against \u003cem\u003eH. armigera\u003c/em\u003e. Although no direct mortality of \u003cem\u003eH. armigera\u003c/em\u003e caterpillars was observed in our study, the relatively short experimental period (9 days) may have limited the detection of longer-term effects.\u003c/p\u003e \u003cp\u003eMaize responds to herbivory by releasing a blend of volatiles that attract natural enemies and prime neighboring plants for defense (Turlings \u0026amp; Ton \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kim \u0026amp; Felton \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Thompson et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Beneficial fungal colonization can influence HIPV emissions (Holopainen \u0026amp; Gershenzon \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pangesti et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fernandez-Conradi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), potentially shaping interactions at higher trophic levels. We investigated whether \u003cem\u003eT. virens\u003c/em\u003e colonization alters maize HIPV emissions induced by \u003cem\u003eH. armigera\u003c/em\u003e feeding. Our study identified a range of volatiles, including monoterpenes, sesquiterpenes, aromatic compounds, and green leaf volatiles (GLVs), primarily released in response to herbivory. However, \u003cem\u003eT. virens\u003c/em\u003e root colonization had little influence on the composition of HIPVs emitted by maize leaves. Despite some variability in individual compounds such as (\u003cem\u003eZ\u003c/em\u003e)-3-hexen-1-ol acetate, neither the wild-type nor the \u003cem\u003evir4\u003c/em\u003e mutant significantly affected the overall HIPV blend.\u003c/p\u003e \u003cp\u003eIn contrast, fungal-induced changes in VOC emissions and indirect plant defenses has been documented in other systems. For example, \u003cem\u003eT. harzianum\u003c/em\u003e enhanced methyl salicylate and β-caryophyllene emissions in aphid infested tomatoes, increasing aphid parasitoid attraction (Coppola et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Similarly, \u003cem\u003eT. harzianum\u003c/em\u003e and \u003cem\u003eT. atroviride\u003c/em\u003e improved maize tolerance to herbivores by modifying volatile terpene emissions that attract predators (Contreras-Cornejo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The fungus \u003cem\u003eT. atroviride\u003c/em\u003e also influenced \u003cem\u003eS\u003c/em\u003e. \u003cem\u003elittoralis\u003c/em\u003e and \u003cem\u003eM. euphorbiae\u003c/em\u003e survival and growth rates via transcriptional changes in defense genes while enhancing parasitoid attraction through altered VOC profiles in tomato, even in the absence of herbivory (Coppola et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). Additionally, \u003cem\u003eFusarium solani\u003c/em\u003e strain K reduced \u003cem\u003eTetranychus urticae\u003c/em\u003e performance in tomato by modifying gene expression and VOC emissions, enhancing \u003cem\u003eM. pygmaeus\u003c/em\u003e attraction (Pappas et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, \u003cem\u003eT. longibrachiatum\u003c/em\u003e increased \u003cem\u003eM. pygmaeus\u003c/em\u003e attraction through HIPV modulation (Battaglia et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, in our study, \u003cem\u003eT. virens\u003c/em\u003e colonization did not significantly alter maize VOC profiles, suggesting that the \u003cem\u003eTrichoderma\u003c/em\u003e effects on tri-trophic interactions may be strain- and host-specific (Rodriguez \u0026amp; Redman \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Y-tube olfactometer assays, \u003cem\u003eM. pygmaeus\u003c/em\u003e exhibited a significant preference for maize HIPVs induced by \u003cem\u003eH. armigera\u003c/em\u003e feeding, but fungal colonization did not further influence predator attraction to maize colonized by the fungal strains. This suggests that \u003cem\u003eT. virens\u003c/em\u003e does not markedly alter VOC profiles in a way that affects third-trophic interactions. Similar findings were reported for \u003cem\u003eT. harzianum\u003c/em\u003e, which also did not significantly modify tomato HIPVs or influence \u003cem\u003eM. pygmaeus\u003c/em\u003e attraction (Meesters et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Equally, fungal-derived volatiles did not affect the indirect defense of \u003cem\u003eBrassica rapa\u003c/em\u003e against \u003cem\u003ePieris brassicae\u003c/em\u003e when plant roots were directly exposed to fungal VOCs, nor did they influence parasitism by \u003cem\u003eCotesia glomerata\u003c/em\u003e or alter the amount or quality of plant-emitted volatiles, resulting in no effects on indirect defense (Moisan et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that \u003cem\u003eT. virens\u003c/em\u003e modulates plant-herbivore interactions but does not significantly affect plant-herbivore-predator dynamics. Our findings highlight the crucial role of fungal sesquiterpenes in enhancing maize resistance against \u003cem\u003eH. armigera\u003c/em\u003e through direct defense mechanisms. To our knowledge, this is the first study to show that \u003cem\u003eT. virens\u003c/em\u003e colonization negatively affects \u003cem\u003eH. armigera\u003c/em\u003e performance via defense responses mediated by the \u003cem\u003evir4\u003c/em\u003e gene cluster. These results underscore the potential of \u003cem\u003eT. virens\u003c/em\u003e in strengthening maize resistance to herbivory and emphasize the need for further research into the genetic and metabolic pathways underlying \u003cem\u003eT. virens\u003c/em\u003e-induced direct defense against \u003cem\u003eH. armigera\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the financial support provided by the Deutsche Akademische Austauschdienst (DAAD) to the first author through the \u0026quot;Research Grants \u0026ndash; Doctoral Programmes in Germany\u0026quot; program. Our sincere thanks go to Jonas Watterott for his assistance with GC-MS analyses. We also thank Christian Ulrich Baden from Bayer AG for providing \u003cem\u003eH. armigera\u003c/em\u003e eggs used in this study. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN.N.:\u003c/strong\u003e Conceptualization, experimental design, experiment execution, data analysis and visualization, methodology, and writing the original manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM.R.:\u003c/strong\u003e Project conception and conceptualization, validation of analyses, methodology, project administration, resource acquisition, supervision, manuscript review, writing, and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.M.:\u003c/strong\u003e Provided resources and supervision, reviewed the manuscript, and contributed revisions and comments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeutsche Akademische Austauschdienst (DAAD) provided a doctoral studies scholarship to the first author through the \u0026quot;Research Grants \u0026ndash; Doctoral Programmes in Germany\u0026quot; program.\u003c/p\u003e\n\u003cp\u003eAll authors have agreed upon the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlfano G, Ivey MLL, Cakir C, et al (2007) Systemic Modulation of Gene Expression in Tomato by \u003cem\u003eTrichoderma hamatum\u003c/em\u003e 382. Phytopathology\u0026reg; 97:429\u0026ndash;437. https://doi.org/10.1094/PHYTO-97-4-0429 \u003c/li\u003e\n\u003cli\u003eAlın\u0026ccedil; T, Cusumano A, Peri E, et al (2021) \u003cem\u003eTrichoderma harzianum\u003c/em\u003e Strain T22 Modulates Direct Defense of Tomato Plants in Response to \u003cem\u003eNezara viridula\u003c/em\u003e Feeding Activity. J Chem Ecol 47:455\u0026ndash;462. https://doi.org/10.1007/s10886-021-01260-3 \u003c/li\u003e\n\u003cli\u003eAlın\u0026ccedil;, T., Peri, E., Torta, L., Guarino, S., Colazza, S., Lievens, B., \u0026amp; Cusumano, A. (2024). Root inoculation with beneficial soil microbes enhances indirect plant defences induced by insect feeding and egg deposition. Functional Ecology. https://doi.org/10.1111/1365-2435.14594 \u003c/li\u003e\n\u003cli\u003eAartsma Y, Bianchi FJJA, Van Der Werf W, et al (2017) Herbivore‐induced plant volatiles and tritrophic interactions across spatial scales. New Phytol 216:1054\u0026ndash;1063. https://doi.org/10.1111/nph.14475 \u003c/li\u003e\n\u003cli\u003eBattaglia D, Bossi S, Cascone P, et al (2013) Tomato Below Ground\u0026ndash;Above Ground Interactions: \u003cem\u003eTrichoderma longibrachiatum \u003c/em\u003eAffects the Performance of \u003cem\u003eMacrosiphum euphorbiae\u003c/em\u003e and Its Natural Antagonists. Mol Plant Microbe Interact 26:1249\u0026ndash;1256. https://doi.org/10.1094/MPMI-02-13-0059-R. \u003c/li\u003e\n\u003cli\u003eBrotman Y, Landau U, Cuadros-Inostroza \u0026Aacute;, et al (2013) \u003cem\u003eTrichoderma\u003c/em\u003e-Plant Root Colonization: Escaping Early Plant Defense Responses and Activation of the Antioxidant Machinery for Saline Stress Tolerance. PLoS Pathog 9:e1003221. https://doi.org/10.1371/journal.ppat.1003221 \u003c/li\u003e\n\u003cli\u003eContreras-Cornejo HA, Mac\u0026iacute;as-Rodr\u0026iacute;guez L, del-Val E, Larsen J (2018) The root endophytic fungus \u003cem\u003eTrichoderma atroviride\u003c/em\u003e induces foliar herbivory resistance in maize plants. Appl Soil Ecol 124:45\u0026ndash;53. https://doi.org/10.1016/j.apsoil.2017.10.004 \u003c/li\u003e\n\u003cli\u003eContreras-Cornejo HA, Mac\u0026iacute;as-Rodr\u0026iacute;guez L, del-Val E, Larsen J (2016) Ecological functions of \u003cem\u003eTrichoderma\u003c/em\u003e spp. and their secondary metabolites in the rhizosphere: interactions with plants. FEMS Microbiol Ecol 92:fiw036. https://doi.org/10.1093/femsec/fiw036 \u003c/li\u003e\n\u003cli\u003eContreras-Cornejo HA, Mac\u0026iacute;as-Rodr\u0026iacute;guez L, Real-Santill\u0026aacute;n RO, et al (2021) In a belowground multitrophic interaction, \u003cem\u003eTrichoderma harzianum\u003c/em\u003e induces maize root herbivore tolerance against \u003cem\u003ePhyllophaga vetula\u003c/em\u003e. Pest Manag Sci 77:3952\u0026ndash;3963. https://doi.org/10.1002/ps.6415 \u003c/li\u003e\n\u003cli\u003eCoppola M, Cascone P, Chiusano ML, et al (2017) \u003cem\u003eTrichoderma harzianum\u003c/em\u003e enhances tomato indirect defense against aphids. Insect Sci 24:1025\u0026ndash;1033. https://doi.org/10.1111/1744-7917.12475 \u003c/li\u003e\n\u003cli\u003eCoppola M, Cascone P, Lelio ID, et al (2019a) \u003cem\u003eTrichoderma atroviride\u003c/em\u003e P1 Colonization of Tomato Plants Enhances Both Direct and Indirect Defense Barriers Against Insects. Front Physiol 10.\u003cu\u003e https://doi.org/doi: 10.3389/fphys.2019.00813 \u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eCoppola M, Diretto G, Digilio MC, et al (2019b) Transcriptome and Metabolome Reprogramming in Tomato Plants by \u003cem\u003eTrichoderma harzianum\u003c/em\u003e strain T22 Primes and Enhances Defense Responses Against Aphids. Front Physiol 10: https://doi.org/10.3389/fphys.2019.00745 \u003c/li\u003e\n\u003cli\u003eDicke M, van Loon JJA, Soler R (2009) Chemical complexity of volatiles from plants induced by multiple attack. Nat Chem Biol 5:317\u0026ndash;324. https://doi.org/10.1038/nchembio.169 \u003c/li\u003e\n\u003cli\u003eEbrahimi M, Mahdian K, De Clercq P (2019) Life‐history parameters and predation capacity of \u003cem\u003eMacrolophus pygmaeus\u003c/em\u003e and Nesidiocoris\u003cem\u003e tenuis\u003c/em\u003e (Hemiptera: Miridae) on eggs of \u003cem\u003ePlutella xylostella\u003c/em\u003e (Lepidoptera: Plutellidae). Agric For Entomol 21:50\u0026ndash;57. https://doi.org/10.1111/afe.12302 \u003c/li\u003e\n\u003cli\u003eFernandez-Conradi P, Jactel H, Robin C, et al (2018) Fungi reduce preference and performance of insect herbivores on challenged plants. Ecology 99:300\u0026ndash;311. https://doi.org/10.1002/ecy.2044 \u003c/li\u003e\n\u003cli\u003eHarman GE (2005) Overview of Mechanisms and Uses of \u003cem\u003eTrichoderma\u003c/em\u003e spp. Phytopathology\u0026reg; 96:190\u0026ndash;194. https://doi.org/10.1094/PHYTO-96-0190 \u003c/li\u003e\n\u003cli\u003eHarman GE, Howell CR, Viterbo A, et al (2004) \u003cem\u003eTrichoderma \u003c/em\u003especies \u0026mdash; opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43\u0026ndash;56. https://doi.org/10.1038/nrmicro797 \u003c/li\u003e\n\u003cli\u003eHermosa R, Viterbo A, Chet I, Monte E (2012) Plant-beneficial effects of \u003cem\u003eTrichoderma\u003c/em\u003e and of its genes. Microbiology 158:17\u0026ndash;25. https://doi.org/10.1099/mic.0.052274-0 \u003c/li\u003e\n\u003cli\u003eHermosa, R., Cardoza, R. E., Rubio, M. B., Guti\u0026eacute;rrez, S., \u0026amp; Monte, E. (2014). Secondary Metabolism and Antimicrobial Metabolites of \u003cem\u003eTrichoderma\u003c/em\u003e. In Biotechnology and Biology of Trichoderma (pp. 125\u0026ndash;137). Elsevier. https://doi.org/10.1016/B978-0-444-59576-8.00010-2 \u003c/li\u003e\n\u003cli\u003eHilker M, Meiners T (2006) Early Herbivore Alert: Insect Eggs Induce Plant Defense. J Chem Ecol 32:1379\u0026ndash;1397. https://doi.org/10.1007/s10886-006-9057-4 \u003c/li\u003e\n\u003cli\u003eHolopainen JK, Gershenzon J (2010) Multiple stress factors and the emission of plant VOCs. Trends Plant Sci 15:176\u0026ndash;184. https://doi.org/10.1016/j.tplants.2010.01.006 \u003c/li\u003e\n\u003cli\u003eIngegno BL, La-Spina M, Jordan MJ, et al (2016) Host Plant Perception and Selection in the Sibling Species \u003cem\u003eMacrolophus melanotoma\u003c/em\u003e and \u003cem\u003eMacrolophus pygmaeus\u003c/em\u003e (Hemiptera: Miridae). J Insect Behav 29:117\u0026ndash;142. https://doi.org/10.1007/s10905-016-9549-1 \u003c/li\u003e\n\u003cli\u003eJim\u0026eacute;nez-Bremont, J. F., Gonz\u0026aacute;lez-P\u0026eacute;rez, E., Ortega-Amaro, M. A., Madrigal-Ortiz, S., Duque-Ortiz, A., \u0026amp; Mendoza-Mendoza, A. (2024). Volatile organic compounds emitted by \u003cem\u003eTrichoderma\u003c/em\u003e: Small molecules with biotechnological potential. Scientia Horticulturae, 325, 112656. https://doi.org/10.1016/j.scienta.2023.112656 \u003c/li\u003e\n\u003cli\u003eKim J, Felton GW (2013) Priming of antiherbivore defensive responses in plants. Insect Sci 20:273\u0026ndash;285. https://doi.org/10.1111/j.1744-7917.2012.01584.x\u003c/li\u003e\n\u003cli\u003eKramer R, Abraham W-R (2012) Volatile sesquiterpenes from fungi: what are they good for? Phytochem Rev 11:15\u0026ndash;37. https://doi.org/10.1007/s11101-011-9216-2 \u003c/li\u003e\n\u003cli\u003eLelio ID, Coppola M, Comite E, et al (2021) Temperature Differentially Influences the Capacity of \u003cem\u003eTrichoderma\u003c/em\u003e Species to Induce Plant Defense Responses in Tomato Against Insect Pests. Front Plant Sci 12:. \u003cu\u003ehttps://doi.org/doi: 10.3389/fpls.2021.678830\u003c/u\u003e \u003c/li\u003e\n\u003cli\u003eLelio ID, Forni G, Magoga G, et al (2023) A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota. 120: https://doi.org/10.1073/pnas.2216922120 \u003c/li\u003e\n\u003cli\u003eLins JC, van Loon JJA, Bueno VHP, et al (2014) Response of the zoophytophagous predators \u003cem\u003eMacrolophus pygmaeus\u003c/em\u003e and \u003cem\u003eNesidiocoris tenuis\u003c/em\u003e to volatiles of uninfested plants and to plants infested by prey or conspecifics. BioControl 59:707\u0026ndash;718. https://doi.org/10.1007/s10526-014-9602-y \u003c/li\u003e\n\u003cli\u003eMac\u0026iacute;as-Rodr\u0026iacute;guez L, Contreras-Cornejo HA, Adame-Garnica SG, et al (2020) The interactions of \u003cem\u003eTrichoderma\u003c/em\u003e at multiple trophic levels: inter-kingdom communication. Microbiol Res 240:126552. https://doi.org/10.1016/j.micres.2020.126552 \u003c/li\u003e\n\u003cli\u003eMarra R, Ambrosino P, Carbone V, et al (2006) Study of the three-way interaction between \u003cem\u003eTrichoderma atroviride\u003c/em\u003e, plant and fungal pathogens by using a proteomic approach. Curr Genet 50:307\u0026ndash;321. https://doi.org/10.1007/s00294-006-0091-0 \u003c/li\u003e\n\u003cli\u003eMaselou DA, Eirini Anastasaki, Panagiotis G. Milonas (2019) The Role of Host Plants, Alternative Food Resources and Herbivore Induced Volatiles in Choice Behavior of an Omnivorous Predator. Front Ecol Evol 6:. https://doi.org/10.3389/fevo.2018.00241 \u003c/li\u003e\n\u003cli\u003eMassalha H, Korenblum E, Tholl D, Aharoni A (2017) Small molecules below-ground: the role of specialized metabolites in the rhizosphere. Plant J 90:788\u0026ndash;807. https://doi.org/10.1111/tpj.13543 \u003c/li\u003e\n\u003cli\u003eMeesters C, Weldegergis BT, Dicke M, et al (2024) Limited effects of plant-beneficial fungi on plant volatile composition and host-choice behavior of \u003cem\u003eNesidiocoris tenuis\u003c/em\u003e. Front Plant Sci. https://doi.org/10.3389/fpls.2023.1322719 \u003c/li\u003e\n\u003cli\u003eMoisan K, Lucas‐Barbosa D, Villela A, et al (2020) No evidence of modulation of indirect plant resistance of Brassica rapa plants by volatiles from soil‐borne fungi. Ecol Entomol 45:1200\u0026ndash;1211. https://doi.org/10.1111/een.12906 \u003c/li\u003e\n\u003cli\u003eMonte E (2023). The sophisticated evolution of \u003cem\u003eTrichoderma\u003c/em\u003e to control insect pests. Proceedings of the National Academy of Sciences, 120(12), e2301971120. https://doi.org/10.1073/pnas.2301971120 \u003c/li\u003e\n\u003cli\u003eMor\u0026aacute;n-Diez ME, Mart\u0026iacute;nez De Alba \u0026Aacute;E, Rubio MB, et al (2021) \u003cem\u003eTrichoderma\u003c/em\u003e and the Plant Heritable Priming Responses. J Fungi 7:318. https://doi.org/10.3390/jof7040318 \u003c/li\u003e\n\u003cli\u003eNawrocka J (2013) Diversity in plant systemic resistance induced by \u003cem\u003eTrichoderma\u003c/em\u003e. Biol Control 67:149\u0026ndash;156. http://dx.doi.org/10.1016/j.biocontrol.2013.07.005 \u003c/li\u003e\n\u003cli\u003eNogueira-Lopez G, Greenwood DR, Middleditch M, et al (2018) The Apoplastic Secretome of \u003cem\u003eTrichoderma virens\u003c/em\u003e During Interaction With Maize Roots Shows an Inhibition of Plant Defense and Scavenging Oxidative Stress Secreted Proteins. Front Plant Sci 9:409. https://doi.org/10.3389/fpls.2018.00409 \u003c/li\u003e\n\u003cli\u003ePangesti N, Pineda A, Pieterse CMJ, Dicke M (2013) Two-way plant-mediated interactions between root-associated microbes and insects: from ecology to mechanisms. Front Plant Sci 4. https://doi.org/0.3389/fpls.2013.00414 \u003c/li\u003e\n\u003cli\u003ePappas ML, Liapoura M, Papantoniou D, et al (2018) The Beneficial Endophytic Fungus \u003cem\u003eFusarium solani\u003c/em\u003e Strain K Alters Tomato Responses Against Spider Mites to the Benefit of the Plant. Front Plant Sci 9:1603. https://doi.org/10.3389/fpls.2018.01603 \u003c/li\u003e\n\u003cli\u003ePieterse CMJ, Zamioudis C, Berendsen RL, et al (2014) Induced Systemic Resistance by Beneficial Microbes. Annu Rev Phytopathol 52:347\u0026ndash;375. https://doi.org/10.1146/annurev-phyto-082712-102340 \u003c/li\u003e\n\u003cli\u003ePineda A, Zheng S-J, van Loon JJA, et al (2010) Helping plants to deal with insects: the role of beneficial soil-borne microbes. Trends Plant Sci 15:507\u0026ndash;514. https://doi.org/10.1016/j.tplants.2010.05.007 \u003c/li\u003e\n\u003cli\u003ePoveda J (2021) Trichoderma as biocontrol agent against pests: New uses for a mycoparasite. Biol Control 159:104634. https://doi.org/10.1016/j.biocontrol.2021.104634 \u003c/li\u003e\n\u003cli\u003eRahman S, Michael Rost\u0026aacute;s, Ilka Vosteen (2024) Drought aggravates plant stress by favouring aphids and weakening indirect defense in a sugar beet tritrophic system. J Pest Sci 1\u0026ndash;16. https://doi.org/10.1007/s10340-024-01799-6 \u003c/li\u003e\n\u003cli\u003eReino, J. L., Guerrero, R. F., Hern\u0026aacute;ndez-Gal\u0026aacute;n, R., \u0026amp; Collado, I. G. (2007). Secondary metabolites from species of the biocontrol agent \u003cem\u003eTrichoderma\u003c/em\u003e. Phytochemistry Reviews, 7(1), 89\u0026ndash;123. https://doi.org/10.1007/s11101-006-9032-2 \u003c/li\u003e\n\u003cli\u003eRodriguez R, Redman R (2008) More than 400 million years of evolution and some plants still can\u0026rsquo;t make it on their own: plant stress tolerance via fungal symbiosis. J Exp Bot 59:1109\u0026ndash;1114. https://doi.org/10.1093/jxb/erm342 \u003c/li\u003e\n\u003cli\u003eSalwan, R., Rialch, N., \u0026amp; Sharma, V. (2019). Bioactive Volatile Metabolites of \u003cem\u003eTrichoderma\u003c/em\u003e: An overview. In H. B. Singh, C. Keswani, M. S. Reddy, E. Sansinenea, \u0026amp; C. Garc\u0026iacute;a-Estrada (Eds.), Secondary Metabolites of Plant Growth Promoting Rhizomicroorganisms (pp. 87\u0026ndash;111). Springer Singapore. https://doi.org/10.1007/978-981-13-5862-3_5 \u003c/li\u003e\n\u003cli\u003eSchausberger P, Peneder S, Jurschik S, Hoffmann D (2012) \u003cem\u003eMycorrhiza\u003c/em\u003e changes plant volatiles to attract spider mite enemies. Funct Ecol 26:441\u0026ndash;449. \u003cu\u003ehttps://doi.org/doi: 10.1111/j.1365-2435.2011. 01947.x\u003c/u\u003e \u003c/li\u003e\n\u003cli\u003eSchweiger R, Padilla-Arizmendi F, Nogueira-L\u0026oacute;pez G, et al (2021) Insights into Metabolic Changes Caused by the \u003cem\u003eTrichoderma virens\u003c/em\u003e\u0026ndash;Maize Root Interaction. Mol Plant-Microbe Interactions\u0026reg; 34:524\u0026ndash;537. https://doi.org/10.1094/MPMI-04-20-0081-R \u003c/li\u003e\n\u003cli\u003eShoresh M, Harman GE (2008) The Molecular Basis of Shoot Responses of Maize Seedlings to \u003cem\u003eTrichoderma harzianum\u003c/em\u003e T22 Inoculation of the Root: A Proteomic Approach. Plant Physiol 147:2147\u0026ndash;2163. https://doi.org/10.1104/pp.108.123810 \u003c/li\u003e\n\u003cli\u003eShoresh M, Harman GE, Mastouri F (2010) Induced Systemic Resistance and Plant Responses to Fungal Biocontrol Agents. Annu Rev Phytopathol 48:21\u0026ndash;43. https://doi.org/10.1146/annurev-phyto-073009-114450 \u003c/li\u003e\n\u003cli\u003eSilva DB, Urbaneja A, P\u0026eacute;rez-Hedo M (2021) Response of mirid predators to synthetic herbivore-induced plant volatiles. Entomol Exp Appl 169:125\u0026ndash;132. https://doi.org/10.1111/eea.12970 \u003c/li\u003e\n\u003cli\u003eThompson MN, Arriaga J, Bradford BJ, et al (2023) Belowground insect herbivory induces systemic volatile emissions that strengthen neighbouring plant resistance aboveground. Plant Cell Environ 47. \u003cu\u003ehttps://doi.org/DOI: 10.1111/pce.14762\u003c/u\u003e\u003cu\u003e \u003c/u\u003e \u003c/li\u003e\n\u003cli\u003eTurlings TCJ, Davison AC, Tam\u0026Ograve; C (2004) A six-arm olfactometer permitting simultaneous observation of insect attraction and odor trapping. Physiol Entomol 29:45\u0026ndash;55. https://doi.org/10.1111/j.1365-3032.2004.0362.x\u003cu\u003e \u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eTurlings TC, Ton J (2006) Exploiting scents of distress: the prospect of manipulating herbivore-induced plant odours to enhance the control of agricultural pests. Curr Opin Plant Biol 9:421\u0026ndash;427. https://doi.org/10.1016/j.pbi.2006.05.010 \u003c/li\u003e\n\u003cli\u003eUrbaneja A, Mont\u0026oacute;n H, Moll\u0026aacute; O (2009) Suitability of the tomato borer \u003cem\u003eTuta absoluta\u003c/em\u003e as prey for \u003cem\u003eMacrolophus pygmaeus\u003c/em\u003e and \u003cem\u003eNesidiocoris tenuis\u003c/em\u003e. J Appl Entomol 133:292\u0026ndash;296. https://doi.org/10.1111/j.1439-0418.2008.01319.x \u003c/li\u003e\n\u003cli\u003eVinale, F., \u0026amp; Sivasithamparam, K. (2020). Beneficial effects of \u003cem\u003eTrichoderma\u003c/em\u003e secondary metabolites on crops. Phytotherapy Research, 34(11), 2835\u0026ndash;2842. https://doi.org/10.1002/ptr.6728 \u003c/li\u003e\n\u003cli\u003eVinale F, Sivasithamparam K, Ghisalberti EL, et al (2008) \u003cem\u003eTrichoderma\u003c/em\u003e\u0026ndash;plant\u0026ndash;pathogen interactions. Soil Biol Biochem 40:1\u0026ndash;10. https://doi.org/10.1016/j.soilbio.2007.07.002 \u003c/li\u003e\n\u003cli\u003eVinci G, Cozzolino V, Mazzei P, et al (2018) An alternative to mineral phosphorus fertilizers: The combined effects of \u003cem\u003eTrichoderma harzianum\u003c/em\u003e and compost on \u003cem\u003eZea mays\u003c/em\u003e, as revealed by 1H NMR and GC-MS metabolomics. PLOS ONE 13:e0209664. https://doi.org/10.1371/journal.pone.0209664 \u003c/li\u003e\n\u003cli\u003eWoo SL, Hermosa R, Lorito M, Monte E (2022) \u003cem\u003eTrichoderma\u003c/em\u003e: a multipurpose, plant-beneficial microorganism for eco-sustainable agriculture. Nat Rev Microbiol 1\u0026ndash;15. https://doi.org/10.1038/s41579-022-00819-5 \u003c/li\u003e\n\u003cli\u003eZeilinger, S., Gruber, S., Bansal, R., \u0026amp; Mukherjee, P. K. (2016). Secondary metabolism in \u003cem\u003eTrichoderma\u003c/em\u003e \u0026ndash; Chemistry meets genomics. Fungal Biology Reviews, 30(2), 74\u0026ndash;90. https://doi.org/10.1016/j.fbr.2016.05.001 \u003c/li\u003e\n\u003cli\u003eZhou D, Huang X-F, Guo J, et al (2018) \u003cem\u003eTrichoderma gamsii\u003c/em\u003e affected herbivore feeding behaviour on \u003cem\u003eArabidopsis thaliana\u003c/em\u003e by modifying the leaf metabolome and phytohormones. Microb Biotechnol 11:1195\u0026ndash;1206. https://doi.org/10.1111/1751-7915.13310 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-chemical-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joce","sideBox":"Learn more about [Journal of Chemical Ecology](https://www.springer.com/journal/10886)","snPcode":"10886","submissionUrl":"https://submission.nature.com/new-submission/10886/3","title":"Journal of Chemical Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Trichoderma virens, Helicoverpa armigera, Macrolophus pygmaeus, maize, tri-trophic interactions, GC-MS, HIPVs, defense priming","lastPublishedDoi":"10.21203/rs.3.rs-6548157/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6548157/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eTrichoderma\u003c/em\u003e species are widely used as root-colonizing biocontrol agents that enhance plant resistance to biotic and abiotic stresses while promoting growth. These fungi produce diverse volatile and non-volatile metabolites that mediate interactions with plants. \u003cem\u003eTrichoderma\u003c/em\u003e can influence both direct and indirect plant defenses, including the release of herbivore-induced plant volatiles (HIPVs) that attract natural enemies of herbivores. In this study, we examined the effects of \u003cem\u003eT. virens\u003c/em\u003e and its \u003cem\u003evir4\u003c/em\u003e gene \u003cem\u003e(\u003c/em\u003eregulating terpenioid synthesis) knockout-mutant on maize (\u003cem\u003eZea mays\u003c/em\u003e), the herbivore \u003cem\u003eHelicoverpa armigera\u003c/em\u003e, and its predator \u003cem\u003eMacrolophus pygmaeus\u003c/em\u003e. Previous research has shown that \u003cem\u003eT. virens\u003c/em\u003e differentially modulate maize root gene expression and specialized metabolite concentrations. Here, we found that caterpillars feeding on maize seedlings colonized by wild-type \u003cem\u003eT. virens\u003c/em\u003e gained significantly less weight than those feeding on maize colonized by the \u003cem\u003evir4\u003c/em\u003e knockout mutant or uncolonized plants, suggesting that the \u003cem\u003evir4\u003c/em\u003e gene cluster contributes to herbivore resistance. Although fungal colonization led to moderate changes in HIPV composition, total volatile emissions remained unchanged. In Y-tube assays, \u003cem\u003eM. pygmaeus\u003c/em\u003e preferred caterpillar-infested maize over healthy plants, but fungal colonization did not significantly affect predator behavior. Our findings demonstrate that \u003cem\u003eT. virens\u003c/em\u003e enhances direct plant defense against herbivores while neutralising indirect defense through a mechanism regulated by terpenoid synthesis depending on \u003cem\u003evir4\u003c/em\u003e gene. Further research is needed to elucidate the metabolic changes in maize induced by \u003cem\u003eT. virens\u003c/em\u003e that contribute to reduced herbivore performance.\u003c/p\u003e","manuscriptTitle":"Sesquiterpene Biosynthetic Gene vir4 from Trichoderma virens Enhances Direct Herbivore Resistance while Maintaining Indirect Defense","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 11:52:08","doi":"10.21203/rs.3.rs-6548157/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-31T15:58:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T13:55:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136895470129279117244545567080269386987","date":"2025-08-21T12:17:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-19T13:55:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198479622588158484469166939149654096282","date":"2025-05-02T15:16:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-02T14:55:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-01T17:01:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-29T12:08:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Chemical Ecology","date":"2025-04-28T12:55:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-chemical-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joce","sideBox":"Learn more about [Journal of Chemical Ecology](https://www.springer.com/journal/10886)","snPcode":"10886","submissionUrl":"https://submission.nature.com/new-submission/10886/3","title":"Journal of Chemical Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f3ec979e-0c6a-448f-911c-f41517622598","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:49:54+00:00","versionOfRecord":{"articleIdentity":"rs-6548157","link":"https://doi.org/10.1007/s10886-025-01681-4","journal":{"identity":"journal-of-chemical-ecology","isVorOnly":false,"title":"Journal of Chemical Ecology"},"publishedOn":"2026-01-13 16:30:51","publishedOnDateReadable":"January 13th, 2026"},"versionCreatedAt":"2025-05-07 11:52:08","video":"","vorDoi":"10.1007/s10886-025-01681-4","vorDoiUrl":"https://doi.org/10.1007/s10886-025-01681-4","workflowStages":[]},"version":"v1","identity":"rs-6548157","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6548157","identity":"rs-6548157","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}