The fungal strains used in this study were F. graminearum (deposit No. CGMCC 3.3488) and T. hamatum (deposit No. CGMCC 20241). The F. graminearum strain was obtained from the China General Microbiological Culture Collection Center (CGMCC), while the T. hamatum strain was originally isolated, purified, and patented by our research group. The strains were initially activated on potato dextrose agar (PDA) plates. The PDA medium was prepared by boiling 200 g of peeled and diced potatoes in 1 L of distilled water for 30 min. The mixture was filtered through cheesecloth, and the filtrate was supplemented with 20 g of glucose (as the dextrose equivalent) and 15 g of agar, then adjusted to a final volume of 1 L with distilled water. The medium was sterilized by autoclaving at 121 °C for 20 min.

The antagonistic activity of T. hamatum (Th) against F. graminearum (Fg) was evaluated using a dual-culture assay on PDA medium, following a previously described method with modifications (Martínez-Padrón et al., 2023). Briefly, a mycelial plug (5 mm in diameter) taken from the actively growing margin of a 3-day-old F. graminearum culture was placed at the center of a fresh PDA plate. Four mycelial plugs of the same size from a 5-day-old T. hamatum culture were then inoculated symmetrically at the four corners of the plate, each approximately 3 cm from the central plug. Control plates were inoculated with only a F. graminearum plug at the center. All plates were sealed with Parafilm and incubated at 25 °C in the dark. The experiment included three independent biological replicates, each consisting of three technical replicate plates (n = 9). The radial growth of F. graminearum toward the T. hamatum colonies was monitored daily. After 5–7 days of incubation, the antagonistic effect was assessed by measuring the inhibition of radial growth in the dual-culture plates compared to the control plates. The percentage inhibition of mycelial growth (PIMG) was calculated as follows: PIMG (%) = [(Rc – Rt) / Rc] × 100, where Rc represents the radial growth of the pathogen in the control plate, and Rt represents the radial growth in the dual-culture plate (Yassin et al., 2021). The interaction zone between the two fungi was also visually examined for signs of mycoparasitism, such as hyphal coiling and lysis.

Plant growth and experimental design. Wheat seeds (Triticum aestivum L. cv. Longmai 35) were sown in plastic pots containing a sterile humus substrate, with 20 seeds per pot (12 pots total). Plants were grown in a controlled climate chamber at 28 °C under a 16/8 h light/dark cycle and 70% relative humidity for 23 days until the seedling stage. The experiment included four treatments: (1) mock-inoculated control (CK, wounded but not inoculated); (2) F. graminearum inoculation (Fg); (3) T. hamatum inoculation (Th); and (4) co-inoculation of F. graminearum and T. hamatum (Th–Fg). Each treatment consisted of three biological replicates, with each replicate comprising three individual seedling stems (nine stems per treatment in total).

Preparation of fungal inocula. F. graminearum and T. hamatum were pre-cultured on PDA medium for 7 days. Mycelial plugs (0.2 cm × 0.2 cm) free of agar were collected from the actively growing margins using a sterile scalpel. Approximately 20 plugs of each fungus were transferred into 300 mL of potato dextrose broth (PDB) and incubated at 28 °C with shaking at 180 rpm for 72 h to form compact mycelial pellets (approximately 0.6 cm in diameter). The pellets were harvested by centrifugation at 5,000 × g for 10 min and washed twice with sterile saline under the same conditions. One mycelial pellet was used to inoculate each wheat seedling.

Seedling inoculation. A longitudinal wound (approximately 1 cm long and less than 50% of the stem diameter in depth) was made on the lowest internode of each seedling using a sterile scalpel. Depending on the treatment, the wound was inoculated with one pellet of Fg, one pellet of Th, or one pellet each of Fg and Th. Control seedlings were wounded but not inoculated.

Disease assessment and sample collection. After inoculation, the treated stem segments were placed on moist filter paper in Petri dishes to maintain humidity and promote disease development. Disease symptoms were monitored daily. Five days post-inoculation, when lesions were visible in Fg-treated plants, a 2-cm stem segment surrounding the inoculation site was excised from each seedling. For each biological replicate, segments from three seedlings were pooled, flash-frozen in liquid nitrogen, and stored at −80 °C for RNA extraction. The entire experiment was repeated independently to ensure reproducibility.

RNA extraction and quality control. Total RNA was extracted from approximately 100 mg of frozen wheat stem segments using the RNAprep Pure Plant Kit (Tiangen, China) according to the manufacturer’s instructions. RNA concentration and purity were measured using a NanoDrop spectrophotometer and a Qubit 2.0 Fluorometer, respectively. RNA integrity was assessed using an Agilent 2,100 Bioanalyzer. Only RNA samples with an OD260/280 ratio of 1.8–2.2, an OD260/230 ratio > 2.0, and an RNA integrity number (RIN) > 7.0 were used for library construction.

Library construction and sequencing. RNA-seq library preparation and sequencing were performed by Beijing Biomarker Technologies Co., Ltd. Briefly, mRNA was isolated from total RNA using oligo(dT)-attached magnetic beads and randomly fragmented. First-strand cDNA was synthesized using random hexamer primers, followed by second-strand synthesis. The double-stranded cDNA was purified with AMPure XP beads, end-repaired, A-tailed, and ligated to adapters. Fragments of 300–400 bp were selected and PCR-amplified to construct the final libraries. Library quality was assessed using an Agilent 2,100 Bioanalyzer and quantified by Qubit 2.0 and qPCR. Libraries with concentrations above 2 nM were sequenced on an Illumina platform NovaSeq 6,000 in paired-end 150 bp (PE150) mode.

Data processing and alignment. Raw paired-end reads were processed with fastp (v0.20.0; Chen et al., 2018) to remove adapters, poly-N sequences, and low-quality bases, yielding high-quality clean data. Clean reads were aligned separately to the following reference genomes using HISAT2 (v2.2.1; Kim et al., 2015): Triticum aestivum (IWGSC RefSeq v2.1; assembly: Triticum_aestivum.v2.1.genome.fa; Plavšin et al., 2021), F. graminearum (strain PH-1; assembly: Fusarium_graminearum_PH-1. ASM24013v3.genome.fa; King et al., 2017), and Trichoderma guizhouense (assembly: Trichoderma_guizhouense. ASM202278v1.genome.fa; Zhu et al., 2021), used as a proxy for T. hamatum due to high genomic similarity.

Transcriptome assembly and quantification. Mapped reads were assembled into transcripts, and their abundances were estimated using StringTie (v2.2.1; Pertea et al., 2015; Shumate et al., 2022) in a reference-based manner. FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) values were calculated by StringTie for transcript-level quantification.

In the equation, cDNA Fragments represents the number of PE reads mapped to the specific transcript; Mapped Fragments (Millions) is the number of all mapped reads, which is counted as 10⁶; Transcript Length(kb) is the length of transcript in unit of 10³b. Differential expression analysis. Read counts per gene were used for differential expression analysis with DESeq2 (v1.30.1; Liu et al., 2021). Genes with an adjusted p-value (FDR) < 0.05 and |log₂(fold change)| > 1 were considered differentially expressed (DEGs). Functional enrichment analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were performed using clusterProfiler (v4.0.5; Xu et al., 2024; Yu, 2024). Terms with a corrected p-value < 0.05 were deemed significantly enriched.

To validate the RNA-seq results, qRT-PCR was performed on 30 differentially expressed genes (DEGs)—10 from T. hamatum, 10 from F. graminearum, and 10 from T. aestivum—selected from the most significantly enriched KEGG pathways in the respective comparisons (e.g., Fg vs. Th–Fg for F. graminearum; Th vs. Th–Fg for T. hamatum; CK vs. Fg/Th/Th–Fg for wheat). The same RNA samples used for RNA-seq were reverse-transcribed into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Japan). qRT-PCR was conducted on a QuantStudio 5 system (Applied Biosystems, United States) with TB Green Premix Ex Taq II (TaKaRa, Japan). Each 20 μL reaction contained 10 μL of TB Green Premix, 0.8 μL each of forward and reverse primers (10 μM), 2 μL of cDNA, and 6.4 μL of nuclease-free water. The thermal profile was: 95 °C for 30 s; 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Melt curve analysis confirmed amplification specificity. All reactions were run in three technical replicates per biological replicate. Primer sequences are listed in Supplementary Table S1. The reference genes used for normalization were actin for T. aestivum, β-tubulin for F. graminearum, and tef1 for T. hamatum. Relative expression was calculated using the 2 ^–ΔΔCt method (Schmittgen and Livak, 2008).

The direct antagonistic capability of T. hamatum against F. graminearum was first evaluated using a dual-culture assay. As depicted in Figure 1, a clear and time-dependent inhibitory effect was observed. In the control plates, F. graminearum grew radially, forming uniform colonies at 3-, 5-, and 7-days post-inoculation (dpi; Figures 1A–C). In stark contrast, the co-culture with T. hamatum resulted in a progressive suppression of the pathogen’s growth. By 5 dpi, a distinct inhibition zone became apparent (Figure 1B), which further expanded by 7 dpi. Quantitative analysis revealed that T. hamatum inhibited the mycelial growth of F. graminearum with an efficacy of 51.1% based on the percentage inhibition of mycelial growth (PIMG), calculated from the radial growth measurements. Notably, at 7 dpi, the interaction zone exhibited a prominent color change to red (Figure 1C), suggesting a severe disruption of the pathogen’s metabolic activity. This result is consistent with previous studies, which have reported significant in vitro antagonism of various Trichoderma species against F. graminearum (Pierson et al., 2025; Mahmoud, 2016). These visual and quantitative results unequivocally demonstrate the potent in vitro antagonistic activity of T. hamatumagainst F. graminearum, providing a phenotypic foundation for the subsequent molecular investigations.

The in planta biocontrol efficacy of T. hamatum was evaluated on wheat seedlings (Figure 2). Over a 7-day period, distinct disease progression was observed across the treatments. Seedlings inoculated with F. graminearum (Fg) alone developed severe necrotic lesions at the inoculation site, which rapidly expanded along the stem. In contrast, seedlings co-inoculated with T. hamatum (Th-Fg) exhibited markedly reduced disease symptoms. The lesions in the Th-Fg treatment were significantly smaller and less severe compared to the Fg-alone treatment at each time point, demonstrating the potent protective effect of T. hamatum. Notably, seedlings mock-inoculated (CK) or inoculated with T. hamatum alone (Th) showed no visible symptoms, confirming that the pathogenicity was solely attributable to F. graminearum and that T. hamatum itself was non-pathogenic to wheat. These results clearly indicate that T. hamatum effectively suppresses disease development caused by F. graminearum in vivo.

To elucidate the transcriptional dynamics underlying the tripartite interaction, we performed RNA-seq on stem samples from all four treatments (CK, Fg, Th, Th-Fg), with three biological replicates per treatment. A total of 12 libraries were constructed and sequenced, generating an average of 54.6 million high-quality clean reads per sample, with Q30 scores exceeding 94.95% and GC contents ranging from 52.47 to 54.52%, indicating high sequencing quality (Supplementary Table S2). A tripartite mapping strategy was employed, wherein the clean reads were separately and simultaneously aligned to the reference genomes of Triticum aestivum(host), F. graminearum (pathogen), and Trichoderma guizhouense (as a proxy for the biocontrol agent, T. hamatum). This approach was chosen to directly and independently capture the concurrent transcriptional responses of all three interacting organisms from the mixed RNA samples, which is essential for dissecting the complex molecular crosstalk within the tripartite system. The clean reads were then separately aligned to the reference genomes of Triticum aestivum, F. graminearum, and Trichoderma guizhouense (as a proxy for T. hamatum). As expected, the vast majority of reads (80.04 to 93.83%) mapped uniquely to the wheat genome across all samples, confirming the plant origin of most transcripts (Supplementary Table S5). Notably, a substantial proportion of reads from the Fg and Th-Fg samples mapped to the F. graminearum genome, confirming successful pathogen colonization (Supplementary Table S3). In contrast, the mapping rate to the T. guizhouense genome was exceptionally low (≤ 0.05%) in all samples, including the Th and Th-Fg treatments (Supplementary Table S4). This suggests that the biomass of T. hamatum in the stem tissues were minimal at the sampling time point, a finding consistent with its role as a rhizosphere-colonizing biocontrol agent rather than an endophyte. The high-quality sequencing data and species-specific mapping efficiency provided a robust foundation for the subsequent differential gene expression analysis.

To dissect the molecular basis of the observed antagonism, we analyzed the transcriptomic response of F. graminearum when challenged by T. hamatum (Fg vs. Th-Fg). The overall gene expression profile, as shown by the FPKM density distribution, indicated a shift in the transcriptome of F. graminearum in the presence of the antagonist (Figure 3A). Comparative analysis identified a total of 889 differentially expressed genes (DEGs; for a complete list, see Supplementary Table S6), with a strong bias toward downregulation (608 genes downregulated vs. 281 upregulated), demonstrating that T. hamatum predominantly suppresses the transcriptional activity of the pathogen (Figure 3B). Functional enrichment analysis of these DEGs provided critical insights into the processes being targeted. GO analysis (Biological Process) revealed that the downregulated genes were significantly enriched in terms related to carbohydrate metabolism, including “polysaccharide catabolic process,” “xylan catabolic process,” and “carbohydrate metabolic process” (Figure 3C). This suggests that T. hamatum impairs the pathogen’s ability to acquire nutrients and maintain cell wall integrity. Furthermore, KEGG pathway analysis showed significant enrichment of DEGs in metabolic pathways such as “Phosphonate and phosphinate metabolism,” “beta-Alanine metabolism,” and “2-Oxocarboxylic acid metabolism” (Figure 3D). Collectively, these transcriptomic findings indicate that the direct antagonistic effect of T. hamatum is mediated, at least in part, by a widespread suppression of key metabolic processes essential for F. graminearum growth and virulence.

In stark contrast to the extensive transcriptional alterations observed in F. graminearum, the transcriptomic profile of T. hamatum itself remained largely stable upon confrontation with the pathogen. The FPKM density distribution showed highly overlapping curves between the Th and Th-Fg treatments, indicating minimal global changes in gene expression (Figure 4A). Comparative analysis (Th vs. Th-Fg) identified only 61 differentially expressed genes (DEGs; see Supplementary Table S7), a remarkably small number, with 60 genes being downregulated and a single gene upregulated (Figure 4B). Functional enrichment analysis of this limited set of DEGs revealed that the most significantly affected biological processes were all related to translation, including “translation,” “translational frameshifting,” and “‘de novo’ cotranslational protein folding” (Figure 4C). Accordingly, KEGG pathway analysis confirmed a significant enrichment in the “Ribosome” pathway (ko03010; Figure 4D). This highly focused transcriptional response, centered on the protein synthesis machinery, suggests that T. hamatum does not undergo a massive metabolic reorganization to combat F. graminearum. Instead, it may rely on pre-formed or constitutively expressed antagonistic machinery, representing a resource-efficient biocontrol strategy that allows for a rapid and energetically favorable response to the pathogen.

We next investigated the impact of F. graminearum infection on the wheat transcriptome. The global gene expression profile revealed a pronounced leftward shift of the FPKM density curve for the Fg treatment compared to the control (CK), indicating a widespread suppression of gene expression in pathogen-inoculated plants (Figure 5A). This massive transcriptional alteration was quantified in the CK vs. Fg comparison, which identified 23,526 differentially expressed genes (DEGs; for a complete list, see Supplementary Table S8), underscoring the profound disruptive effect of the pathogen (Figure 5B). GO enrichment analysis showed that these DEGs were significantly involved in critical biological processes, including the biosynthesis of defense-related compounds like cinnamic acid, carbohydrate metabolism, and cell wall macromolecule catabolism (Figure 5C). KEGG pathway analysis further demonstrated that the infection significantly affected pathways central to plant physiology, such as “Biosynthesis of amino acids,” “Carbon metabolism,” “Carbon fixation in photosynthetic organisms,” and “Glutathione metabolism” (Figure 5D). These findings indicate that F. graminearum infection imposes severe physiological stress on wheat, repressing fundamental metabolic activities including photosynthesis and energy production, while simultaneously triggering complex but potentially dysregulated defense responses.

To determine whether T. hamatum alone could induce a defense response in wheat, we analyzed the transcriptome of plants inoculated only with the biocontrol agent (CK vs. Th). This comparison identified 11,711 differentially expressed genes (DEGs; see Supplementary Table S9), indicating that T. hamatum inoculation significantly reprograms the wheat transcriptome even in the absence of F. graminearum. Functional enrichment analysis of these DEGs revealed a distinct pattern compared to the pathogen response. GO terms significantly enriched were related to “carbohydrate metabolic process,” “cinnamic acid biosynthetic process,” and “L-phenylalanine catabolic process” (Figure 6A). More importantly, KEGG pathway analysis showed a strong activation of well-characterized defense signaling and biosynthesis pathways, most notably “Phenylpropanoid biosynthesis,” “Glutathione metabolism,” and “alpha-Linolenic acid metabolism” (Figure 6B). The upregulation of phenylpropanoid biosynthesis is associated with the production of antimicrobial compounds like flavonoids and lignin, while glutathione and alpha-linolenic acid metabolism are integral to redox homeostasis and jasmonic acid signaling, respectively. These results demonstrate that T. hamatum acts as a potent priming agent, pre-activating a broad spectrum of the wheat defense arsenal and preparing the plant for a more efficient response to subsequent pathogen challenge.

The transcriptomic profile of wheat under dual inoculation (Th-Fg) revealed the integrated outcome of the tripartite interaction. Comparison with the control (CK vs. Th-Fg) identified 14,958 differentially expressed genes (DEGs; for a complete list, see Supplementary Table S10). Strikingly, the functional characteristics of these DEGs differed markedly from those induced by F. graminearum alone. GO enrichment analysis highlighted the significant upregulation of processes including “cinnamic acid biosynthetic process,” “L-phenylalanine catabolic process,” and, most notably, “photosynthesis” (Figure 7A). This indicates that the presence of T. hamatum not only maintained the activation of defense-related pathways but also counteracted the pathogen’s suppression of core physiological functions. KEGG pathway analysis further supported this conclusion, showing significant enrichment in “Carbon fixation in photosynthetic organisms,” “Carbon metabolism,” and “Biosynthesis of amino acids” (Figure 7B). The enhanced expression of genes in these pathways suggests that T. hamatum effectively mitigates the severe physiological damage caused by F. graminearum, helping to restore the plant’s energy production and primary metabolism. Concurrently, the sustained enrichment of phenylpropanoid-related terms points to a robust, ongoing defense activation. These results demonstrate that T. hamatum orchestrates a protective transcriptional reprogramming in wheat that simultaneously reinforces defense capabilities and promotes physiological recovery from pathogen stress.

To validate the reliability of our RNA-seq data, we performed qRT-PCR analysis on 30 differentially expressed genes (DEGs)—10 from T. aestivum, 10 from F. graminearum, and 10 from T. hamatum—selected from the most significantly enriched pathways, as detailed in section 3.4–3.7. The selected wheat genes were representative of the key processes identified in the transcriptome: "defense activation” genes (e.g., pathogenesis-related (PR) genes and phenylpropanoid biosynthesis enzymes) and “stress response” genes (e.g., those encoding heat shock proteins (HSPs) and antioxidant enzymes). As shown in Figure 8, the relative expression trends of the selected DEGs measured by qRT-PCR were in strong agreement with the RNA-seq results across all three comparative groups: CK vs. Fg (Figure 8A), Fg vs. Th-Fg (Figure 8B), and Th vs. Th-Fg (Figure 8C). A significant positive correlation (R² > 0.85, p < 0.001) was observed between the qRT-PCR and RNA-seq datasets. This high consistency robustly confirms the accuracy and reliability of our transcriptomic analysis, and specifically validates the expression patterns of genes central to wheat defense priming and stress mitigation. This provides a solid foundation for the subsequent mechanistic interpretations.

Our study found that F. graminearum exhibited widespread transcriptional suppression when co-inoculated with T. hamatum (Th-Fg), with 608 downregulated DEGs significantly enriched in carbohydrate metabolic processes such as polysaccharide catabolic process and xylan catabolic process (Figure 3C). This finding provides strong molecular evidence for the direct antagonistic action of Th, corroborating previous observations that Trichoderma species can inhibit F. graminearum through direct interactions (Li Z. et al., 2020; Kostrzewska-szlakowska and Kiersztyn, 2017). Carbohydrate metabolism is central to nutrient acquisition, cell wall integrity, and energy generation for fungal pathogens (Zhang et al., 2022). The inhibition of these pathways, including those involving glycosyltransferases (GT), glycoside hydrolases (GH), and polysaccharide lyases (PL) families which are essential for fungal survival and host interaction (Trinca et al., 2023), likely directly contributed to the restricted mycelial growth of Fg observed in the dual-culture assay (Figure 1) and the reduced disease symptoms in planta (Figure 2). This aligns with the known efficacy of Trichoderma asperellum and T. hamatum in inhibiting Fusarium growth through mechanisms such as secreting cell wall-degrading enzymes (Li et al., 2020; Ji et al., 2023; Rabuske et al., 2024). However, our transcriptomic data reveal the global nature of this suppression, suggesting that Th′s attack targets a broad spectrum of fundamental metabolic networks in Fg—potentially disrupting glucose uptake, glycolysis, and the pentose phosphate pathway vital for energy production rather than a single pathway. This multifaceted approach extends beyond the action of specific enzymes, such as the cellulase synthesized by T. koningii (Pang et al., 2021; Xiang et al., 2021), indicating a more comprehensive antagonistic strategy. The downregulation of carbohydrate metabolism genes could also lead to cascading effects on interconnected cellular processes like amino acid and lipid metabolism, potentially causing a systemic failure of essential metabolic functions in the pathogen (Li et al., 2020). This broad-spectrum disruption of central metabolism likely represents a more efficient and durable antagonistic strategy less prone to resistance development, as it simultaneously undermines the pathogen’s capacity to overcome host defenses, which themselves involve complex carbohydrate metabolism pathways (Tu et al., 2023; Eldakak et al., 2018). The effectiveness of this strategy highlights the potential of T. hamatum as a robust biocontrol agent against F. graminearum, a major threat to cereal crops (Tu et al., 2023; Liu et al., 2022).

Strikingly, in contrast to the “transcriptional storm” experienced by the pathogen, T. hamatum itself displayed minimal transcriptional reprogramming when confronting F. graminearum (only 61 DEGs in Th vs. Th-Fg), and these genes were primarily enriched in translation-related functions (Figure 4). This finding suggests a strategic reliance on constitutively expressed antagonistic machinery rather than a massive gene activation response. This minimal transcriptional change implies an evolutionary adaptation where T. hamatum may employ a rapid, pre-emptive defense system against F. graminearum without incurring the high metabolic cost of extensive de novo gene expression and protein synthesis. This observation aligns with the well-documented role of Trichoderma spp. as effective biocontrol agents against a wide range of plant pathogens, including Fusarium spp. (Feng et al., 2025; Pedrero-Méndez et al., 2025; Karuppiah et al., 2022; Ren et al., 2025; Saravanakumar et al., 2017). Their antagonistic activities, which encompass mycoparasitism, competition, and antibiosis (Yao et al., 2023; Priyashantha et al., 2023), may rely heavily on pre-formed or constitutively produced effectors such as cell wall-degrading enzymes and antibiotics. Specifically, the potent antagonistic effects of T. hamatum against various Fusarium species, including F. graminearum as observed in our and others’ confrontation assays (Feng et al., 2025; Ren et al., 2025; Oviya et al., 2022), can therefore be interpreted as a resource-efficient antagonistic strategy. By maintaining a state of constant readiness, T. hamatum can rapidly and effectively suppress pathogen growth, representing a highly efficient biocontrol mechanism.

The second major finding of this study is that T. hamatum alone significantly primes the wheat defense system, inducing a “defense-ready” state characteristic of Induced Systemic Resistance (ISR; Wu et al., 2025; Siddaiah et al., 2017; Dalio et al., 2020). In the absence of the pathogen (CK vs. Th), Th inoculation induced 11,711 wheat DEGs significantly enriched in key defense pathways (Figure 6). The activation of phenylpropanoid biosynthesis, a cornerstone pathway for producing antimicrobial compounds like lignin, phenolics, and flavonoids (Wu et al., 2025; Iriti and Faoro, 2009; Ye et al., 2020), suggests enhanced physical and chemical barrier formation (Yang et al., 2023; Banerjee and Gangopadhyay, 2024). Concurrently, the enrichment of glutathione metabolism indicates an enhanced capacity for scavenging reactive oxygen species (ROS) and maintaining redox homeostasis upon pathogen challenge (Wu et al., 2025; Min et al., 2024; Xu et al., 2019; Wang et al., 2023). Furthermore, the upregulation of alpha-linolenic acid metabolism, a precursor pathway for jasmonic acid (JA) biosynthesis (Min et al., 2024; Jiang et al., 2023), points to a preparedness for JA-mediated defense signaling, which is crucial against necrotrophic pathogens like F. graminearum (Wu et al., 2025; Banerjee and Gangopadhyay, 2024; Tian et al., 2024). This multi-faceted priming effect means wheat can mount a faster and stronger defense response upon actual F. graminearum invasion.

Our findings corroborate that various Trichoderma strains induce plant resistance, but they clearly delineate the specific pathways activated in the FHB pathosystem. It is noteworthy that while F. graminearum infection alone (CK vs. Fg) also triggered some defense-related pathways, it occurred alongside a global suppression of gene expression (Figure 5A), implying a potentially dysregulated defense system due to the pathogen’s virulence strategies (Balsells-Llauradó et al., 2020). In contrast, priming by T. hamatum prior to stress challenge positions wheat in a more advantageous “alerted” state, enabling a more effective and coordinated defense response. This mechanism, involving intricate crosstalk between defense pathways, underscores the potential of T. hamatum as a sustainable strategy to enhance crop resilience against FHB.

Beyond direct pathogen suppression and defense priming, the third innovative finding of this study is Th′s significant role in mitigating the physiological damage caused by Fg to wheat. The most compelling evidence comes from the comparison between the dual inoculation (Th-Fg) and the control (CK). In stark contrast to the general suppression of genes related to photosynthesis and carbon metabolism observed under Fg infection alone (Figure 5D), these crucial physiological processes, such as “photosynthesis,” “carbon fixation in photosynthetic organisms,” and “carbon metabolism,” were significantly enriched and exhibited enhanced expression in the Th-Fg treatment (Figure 7). This indicates that the presence of Th not only contained pathogen growth but, more importantly, helped wheat maintain or restore its fundamental physiological functions. Our findings reveal that the biocontrol efficacy of T. hamatum extends beyond conventional “disease resistance” into the realm of “tolerance” or “health maintenance.” Crucially, T. hamatum mitigates the severe physiological damage caused by F. graminearum (Fg), particularly its suppression of photosynthesis. Photosynthesis, as the fundamental source of energy and biomass, is critical for plant health and minimizing yield loss (Li et al., 2020). By alleviating the pathogen-induced suppression of photosynthetic and carbon metabolism pathways (Figure 7), T. hamatum enables wheat to maintain energy production and resource allocation toward defense and repair processes. This mechanism of enhancing the plant’s intrinsic ability to withstand pathogen pressure—tolerance—differs fundamentally from the mode of action of fungicides that directly kill pathogens (Cheng et al., 2014). The ability of T. hamatum to improve plant health likely involves a combination of effects, including reducing pathogen load (Li et al., 2020) and strengthening overall physiological resilience through mechanisms such as enhanced antioxidant systems (Vindas-Reyes et al., 2024; Tan et al., 2024). This approach aligns with the growing importance of sustainable strategies that complement breeding for resistance (Chu et al., 2011; Gyawali et al., 2023; Cai et al., 2005) and move beyond sole reliance on chemical controls (Powell et al., 2024). By fostering plant tolerance through physiological improvement, T. hamatum represents a promising component of integrated management strategies against Fusarium head blight.