The process of epithelial cell death in Pinus thunbergii caused by the pine wood nematode, Bursaphelenchus xylophilus

This study describes a new technique to investigate how the pine wood nematode (PWN), Bursaphelenchus xylophilus, kills pine epithelial cells. After inoculating PWN into 20-cm-long Pinus thunbergii stem cuttings and incubating for 1, 3 or 7 days, the cuttings were split into 2.5 cm segments. The segments were tangentially cut so that the epithelia of several cortical resin canals were exposed, and these were stained with Evans Blue for the detection of dead epithelial cells. While almost no dead epithelial cells were found in the cortical resin canals of non-PWN-inoculated control cuttings up to day 7 of the experiment, dead epithelial cells were distributed sparsely in the epithelium of cortical resin canals throughout pine cuttings inoculated with PWN 1, 3 and 7 days after inoculation. The sparse and sporadic distribution of dead pine cells in the epithelium suggested that individual PWN attacked one epithelial cell at a time with its stylet and migrated between attacks.