Mechanistic elucidation of a terpenoid nano-bionematicide for the management of root-knot nematodes, Meloidogyne incognita infecting tomato

Plant-parasitic nematodes, particularly Meloidogyne incognita, represent a major constraint to global vegetable production and cause substantial yield losses. Although azadirachtin exhibits strong nematicidal potential, its practical application is limited by instability under field conditions. In this study, a terpenoid-based azadirachtin nano-formulation (Terpaz®, TNF) was developed and evaluated through an integrated approach encompassing physicochemical characterization, in vitro bioassays, molecular docking and molecular dynamics simulations, enzyme inhibition assays, root invasion studies, field evaluation, and biosafety assessment. The characterisation of TNF exhibited a nanoscale droplet size (79.69 nm) with narrow PDI (0.17). In vitro assays revealed substantial inhibition of egg hatching (up to 91.3%) and juvenile mortality (up to 88%), outperforming the synthetic nematicide fluopyram at equivalent doses. Molecular docking demonstrated a strong binding affinity of azadirachtin to acetylcholinesterase (AChE, − 5.37 kcal/mol) and ATPase (–4.8 kcal/mol), which was further supported by stable molecular dynamics trajectories. Enzyme assays confirmed dose-dependent inhibition of AChE (65.7%) and ATPase (73.4%) activities, validating the dual-target mechanism. Root penetration studies showed that TNF at 5 mL/L prevented juvenile invasion beyond the epidermis. Field trials demonstrated that TNF significantly suppressed nematode population densities (Pf = 1.5 J2/g soil), reduced galling, and enhanced tomato yield (25,400 kg/acre), outperforming the synthetic nematicide. Importantly, TNF showed no inhibitory effects on beneficial soil microbes. Collectively, this study establishes TNF as an effective, environmentally safe, and multi-targeted bionematicide, offering a promising sustainable alternative for M. incognita management in horticultural crops.