| Summary: | In the present study, <i>Paenibacillus dendritiformis</i>, <i>Trichoderma harzianum,</i> and <i>Trichoderma asperellum</i> were appraised as potential biocontrol agents that induce resistance in chilli (<i>Capsicum annuum</i>) against the devastating pathogen <i>Colletotrichum truncatum,</i> which causes anthracnose. Bright-field and scanning electron micrographs showed the hyphal degradation, lysis, and abnormal swelling in <i>C. truncatum</i> against <i>P. dendritiformis</i> in a dual plate assay. Under greenhouse conditions, chilli seeds pretreated with <i>P. dendritiformis, T. asperellum, T. harzianum,</i> and <i>T. asperellum</i> + <i>T. harzianum</i> by soil soak method inflicted an induced systemic resistance (ISR) in chilli against a <i>C. truncatum</i>-challenged condition. In chilli, the disease index percentage was significantly reduced in the <i>T. asperellum</i> + <i>T. harzianum</i>-treated seeds, followed by the <i>T. harzianum-</i>, <i>T. asperellum-,</i> and <i>P. dendritiformis</i>-treated seeds as compared to the untreated and challenged, respectively. Chilli seeds were primed with <i>T. asperellum</i> + <i>T. harzianum</i> (78.67%), which revealed maximum disease protection under the challenged condition, followed by <i>T. harzianum</i> (70%), <i>T. asperellum</i> (64%), and <i>P. dendritiformis</i> (56%) as compared to untreated and <i>C. truncatum</i>-challenged (6%) condition served as control. The seeds that were pretreated with biocontrol agents (BCAs) inflicted ISR against <i>C. truncatum</i> by enhancing the activity of defence-related enzymes (superoxide dismutase (SOD), peroxidase (POX), polyphenol oxidase (PPO), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and phenylalanine ammonia-lyase (PAL)), accumulating phenolic compounds, and increasing the relative chlorophyll content in chilli. Nitroblue tetrazolium (NBT) and 3,3′-Diaminobenzidine (DAB) stains were used to detect the accumulation of superoxide anion and hydrogen peroxide that appeared nearby the fungal infection sites. The accumulation of reactive oxygen species (O<sub>2</sub><sup>−</sup> and H<sub>2</sub>O<sub>2</sub>) in the pathogen-inoculated leaves was a maximum of 48 hpi, followed by <i>P. dendritiformis, T. asperellum, T. harzianum,</i> and <i>T. asperellum</i> + <i>T. harzianum</i> treated tissue upon <i>C. truncatum</i>-challenged condition as compared to the control. Overall, our results showed the potential of <i>T. harzianum, T. asperellum, and P. dendritiformis</i> as biocontrol agents that prevent infection by <i>C. truncatum</i> and inflict an induced systemic resistance in chilli by enhancing the biosynthesis of phenolic compounds, defence and antioxidative enzymes, and reducing the lesion development and reactive oxygen species accumulation. This is the first report of induced systemic resistance against anthracnose in chilli obtained by application of <i>T. harzianum</i>, <i>T. asperellum</i> and <i>P. dendritiformis</i>, through seed priming.
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