Antagonistic activity of Pochonia chlamydosporia against three helminth eggs and characterization of its serine protease.

Abstract

To address the economic burden caused by livestock parasitic diseases, particularly gastrointestinal nematodes (GIN) and liver flukes, which are exacerbated by growing anthelmintic resistance, researchers are increasingly focusing on biological control strategies as a promising solution. Among these, the fungus Pochonia chlamydosporia has demonstrated promising helminth control properties. This study explored the potential of P. chlamydosporia in controlling helminth infections by examining its effects on helminth eggs. P. chlamydosporia was cultured on 2 % water agar (WA) plates, and the eggs of three parasite species (Fasciola hepatica, Parascaris spp., and Nematodirus oiratianus) were placed on these plates. The impact of the fungus on the eggs was assessed using light microscopy and scanning electron microscopy (SEM). Eggs were introduced into a liquid medium to stimulate P. chlamydosporia' s predatory activity. The culture filtrate was tested for protease activity and its efficacy against nematode eggs was evaluated. The extracellular alkaline serine protease was purified and characterized through ammonium sulfate precipitation and Sephadex G - 100 chromatography. P. chlamydosporia showed type 1, type 2, and type 3 effects on eggs. (Type 1 effect: physiological and biochemical impact without morphological damage to the eggshell, with visible hyphae adhering to the eggshell; Type 2 effect: lytic effect causing morphological changes in both the embryo and eggshell, without hyphal penetration; Type 3 effect: lytic effect with morphological changes in the embryo and eggshell, along with hyphal penetration and internal egg colonization). Light microscope and SEM observations revealed that P. chlamydosporia destroyed the eggs through mycelial growth, appressoria formation, penetration, and degradation stages. Moreover, the addition of nematode eggs stimulated the secretion of extracellular proteins, including proteases, with induction filtrate showing high ovicidal activity. The molecular mass of the protease was approximately 40 kDa estimated by SDS-PAGE. The optimum activity of the protease was at pH 10 and 60 ℃. The purified protease was highly sensitive to phenylmethyl sulfonyl fluoride (PMSF), indicating it belonged to the serine protease family. The findings suggest that P. chlamydosporia could be an effective biological control agent for helminth diseases in livestock.