Characterization and Biofungicide Potential of a Novel Antifungal defensin, K4CBP6, from Solanum lycopersicum L

The global rise in resistance to chemical fungicides and their strict regulation by the EU, has created an urgent need for alternative antifungal strategies in agriculture. Plant defensins represent promising alternatives owing to their broad-spectrum antifungal activity, structural stability, and low toxicity to mammalian cells and plants. In this study, we identified and characterized a novel antifungal defensin, K4CBP6, from Solanum lycopersicum L., along with its γ-core peptide derivatives, K4CBP6γ1 and K4CBP6γ2, as potential biofungicide agents. Protein database mining revealed a widespread distribution of K4CBP6 homologs within the Solanaceae family. Recombinant K4CBP6 (rK4CBP6) was successfully produced using a Komagataella phaffii-based expression system, while K4CBP6γ1 and K4CBP6γ2 were chemically synthesized. Structural analyses via electrospray ionization mass spectrometry and electronic circular dichroism spectroscopy confirmed a cysteine-stabilized α-helix β-strand folded structure for rK4CBP6. In vitro susceptibility assays demonstrated that both rK4CBP6 and K4CBP6γ2 exhibited antifungal activity against major tomato pathogens, Botrytis cinerea, Cladosporium herbarum, and Fusarium oxysporum with minimum inhibitory concentrations ranging from 12.5 to 25 µg ml^-1. Furthermore, neither rK4CBP6 nor K4CBP6γ2 exhibited cytotoxic effects on mammalian cell lines or adverse effects in animal and plant model systems even at concentrations of 200 and 400 µg ml^-1. Proof-of-concept experiments on tomato plants and fruits confirmed their protective efficacy against B. cinerea and C. herbarum. These findings highlight the potential of rK4CBP6 and K4CBP6γ2 as sustainable biofungicide candidates for plant disease management, owing to their antifungal efficacy both in vitro and in planta, along with their lack of cytotoxic effects.