| Summary: | <i>Trichoderma</i> is a filamentous fungus that is widely distributed in nature. As a biological control agent of agricultural pests, <i>Trichoderma</i> species have been widely studied in recent years. This study aimed to understand the inhibitory mechanism of <i>Trichoderma virens</i> ZT05 on <i>Rhizoctonia solani</i> through the side-by-side culture of <i>T. virens</i> ZT05 and <i>R. solani</i>. To this end, we investigated the effect of volatile and nonvolatile metabolites of <i>T. virens</i> ZT05 on the mycelium growth and enzyme activity of <i>R. solani</i> and analyzed transcriptome data collected from side-by-side culture. <i>T. virens</i> ZT05 has a significant antagonistic effect against <i>R. solani</i>. The mycelium of <i>T. virens</i> ZT05 spirally wraps around and penetrates the mycelium of <i>R. solani</i> and inhibits the growth of <i>R. solani</i>. The volatile and nonvolatile metabolites of <i>T. virens</i> ZT05 have significant inhibitory effects on the growth of <i>R. solani</i>. The nonvolatile metabolites of <i>T. virens</i> ZT05 significantly affect the mycelium proteins of <i>R. solani</i>, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), selenium-dependent glutathione peroxidase (GSH-Px), soluble proteins, and malondialdehyde (MDA). Twenty genes associated with hyperparasitism, including extracellular proteases, oligopeptide transporters, G-protein coupled receptors (GPCRs), chitinases, glucanases, and proteases were found to be upregulated during the antagonistic process between <i>T. virens</i> ZT05 and <i>R. solani</i>. Thirty genes related to antibiosis function, including tetracycline resistance proteins, reductases, the heat shock response, the oxidative stress response, ATP-binding cassette (ABC) efflux transporters, and multidrug resistance transporters, were found to be upregulated during the side-by-side culture of <i>T. virens</i> ZT05 and <i>R. solani</i>. <i>T. virens</i> ZT05 has a significant inhibitory effect on <i>R. solani</i>, and its mechanism of action is associated with hyperparasitism and antibiosis.
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