Nematotoxicity of a Cyt-like protein toxin fromConidiobolus obscurus(Entomophthoromycotina) on the pine wood nematodeBursaphelenchus xylophilus

BACKGROUND The pine wood nematodeBursaphelenchus xylophilusis a destructive pest onPinustrees and lacks effective control measures. The present study identified a novel nematotoxic cytolytic (Cyt)-like protein originating from the entomopathogenic fungusConidiobolus obscurus. RESULTS The protein was successfully purified using heterologous expression inEscherichia coliand affinity chromatography.N-hydroxysuccinimide-rhodamine-labeled Cyt-like protein was used to establish the route of toxin uptake, and revealed that the toxin can enter the nematode via the stylet. In bioassays, the purified protein had high nematicide activity againstB. xylophilus, with a median lethal concentration at 24 h of 15.8 and 29.4 mu g mL(-1)for juveniles and adults, respectively. Compared with the deionized water control, fecundity, thrashing, and egg hatching were significantly reduced by 97%, 98%, and 83%, respectively, with 40 mu g mL(-1)Cyt-like protein at 24-36 h. Staining with Oil-Red-O showed a decrease in large lipid droplet formation in the protein-treated adult nematodes. CONCLUSION The Cyt-like protein toxin possesses high nematicide activity againstB. xylophiluswith effects on nematode vitality and fecundity. The potential exists to use the Cyt-like protein for the control ofB. xylophilus.

Automated summary

A novel nematotoxic cytolytic (Cyt)-like protein was successfully purified from the entomopathogenic fungus Conidiobolus obscurus via heterologous expression in Escherichia coli. This protein showed high nematicide activity against B. xylophilus, significantly impacting the nematode's vitality and fecundity, suggesting its potential for controlling B. xylophilus.