Pre-penetration events in fungal parasitism of nematode eggs

The present investigation deals with the main factors involved in early infection of nematode eggs by fungal parasites. We studied the effect of hydrophobicity on appressorium formation by germlings of Pochonia rubescens (syn. Verticillium suchlasporium), P. chlamydosporia (syn. V. chlamydosporium) and Lecanicillium lecanii (syn. V. lecanii). Appressoria were frequently formed on hydrophobic surfaces such as polyvinyl chloride or polystyrene and were infrequently formed on hydrophilic materials such as glass or aluminium. Infected eggs probed with the FITC-labelled lectin Concanavalin-A showed intense labelling corresponding to appressoria formed by fungal parasites on the eggshell surface. Proteolytic activity was found in extracts from conidia and germlings of fungal parasites (especially P. chlamydosporia) in the absence of nematode eggs. Addition of the serine proteinase inhibitors phenylmetylsulphonyl fluoride (PMSF) or diisopropyl fluorophosphonate (DFP) to the extracts reduced their proteolytic activity. PMSF was the most effective inhibitor. Zymography also revealed proteolytic activity in extracts from the three fungi tested. This activity mostly corresponded to bands of Rf's of substrate degradation similar to that Of purified main protease (P32) from P. rubescens. Other bands with molecular weight higher than P32 (low Rf) were found especially for P. chlamydosporia extracts. For L. lecanii only bands of low Rf were found. Serum anti-P32 partially inhibited proteolytic activity of extracts from conidia and germlings. Application of PMSF and DFP to the inoculum, reduced egg penetration for the three species studied. PMSF caused the highest reduction in eggs infected by L. lecanii, while DFP significantly reduced egg infection by both P. chlamydosporia and L. lecanii. Our results therefore show hydrophobicity, appresorium formation and protease production as factors involved in early parasitism of nematode eggs.