Development of a Trichoderma–Bacillus biofilm inoculant for plant growth promotion and biocontrol of Sclerotium and Fusarium in chickpea

Chickpea (Cicer arietinum) productivity is significantly hindered by soil-borne pathogens such as Fusarium oxysporum f.sp. ciceri (FOC) and Sclerotium rolfsii (SR), which cause wilt and collar rot, respectively. This study aimed to develop a dual-species biofilm formulation using Trichoderma harzianum as a fungal mycelium matrix and Bacillus subtilis as a bacterial partner, to enhance plant growth and provide biocontrol efficacy under pathogen pressure. Biofilm formation was optimized using six media, with Z-medium yielding the highest biomass (4.62 ± 0.10 g fresh weight; 0.42 ± 0.03 g dry weight). Microscopic analyses, including SEM and CSLM, confirmed robust colonization of fungal hyphae by B. subtilis within the biofilm matrix. The biofilm formulation demonstrated superior plant growth-promoting (PGP) traits such as elevated IAA production (22.14 ± 2.6 µg/mL) and ammonia release compared to monocultures. In vitro antifungal assays showed 60% inhibition of pathogen growth by the biofilm, outperforming individual strains. Pot experiments under net-house conditions revealed significant improvements in shoot and root length and reductions in disease incidence. Notably, treatment T4 (FOC + SR + Biofilm) recorded only 19.4% disease incidence compared to 78.3% in the untreated pathogen control. Furthermore, biofilm-treated plants exhibited markedly higher activities of defense-related enzymes PAL, POX, and PPO indicating induced systemic resistance (ISR). The T. harzianum–B. subtilis biofilm formulation demonstrated synergistic effects in promoting plant growth, suppressing disease, and priming host immunity, suggesting its strong potential for sustainable chickpea cultivation under biotic stress conditions.