Nematicidal Activity of Penicillium citrinum Culture Filtrate Against Caenorhabditis elegans and Characterization of Its Extracellular Proteolytic Activity

Extracellular fungal proteases are promising biomolecules with diverse biotechnological applications, including potential roles in biological control of nematodes. The growing demand for sustainable alternatives to chemical nematicides has intensified interest in microbial biocontrol agents for environmentally friendly pest management. In this study, we evaluated the nematicidal activity of Penicillium citrinum culture filtrate against Caenorhabditis elegans and characterized its extracellular proteolytic activity. The culture filtrate exhibited maximum proteolytic activity at 60 °C and pH 9.0, and the activity was inhibited by PMSF, suggesting the presence of PMSF-sensitive proteases. K+, Mg2+, and Mn2+ enhanced proteolytic activity, whereas Zn2+ and Cu2+ showed inhibitory effects. Treatment of C. elegans with the culture filtrate resulted in approximately 60% inhibition of egg hatching after 24 h, 100% larval mortality after 48 h, 94% impairment of larval development to adulthood, 90% reduction in brood size, and 80% reduction in adult survival after 5 days of treatment, with statistically significant differences relative to the untreated control. In all nematicidal assays, PMSF-treated culture filtrate showed markedly reduced activity, indicating a contributory role of PMSF-sensitive proteases. LC-MS/MS analysis of the ammonium sulfate-fractionated culture filtrate detected peptide sequences matching several extracellular proteins, including proteases. Together, these findings demonstrate that the nematicidal activity of P. citrinum culture filtrate indicates that PMSF-sensitive proteolytic activity may contribute to the observed effects against C. elegans. Because the culture filtrate is a complex mixture containing multiple extracellular components, the specific contribution of individual enzymes cannot be established from the present study. Further studies using purified or individually characterized components are required to determine their direct roles and biocontrol potential.