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

Abstract

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.

Migration and Attacking Ability of Bursaphelenchus mucronatus in Pinus thunbergii Stem Cuttings

“…Since Evans blue stains only dead cells ( Gaff and Okong’o-Ogora, 1971 ), any blue-stained cells in the epithelia were regarded as dead cells. The number of dead cells in the exposed epithelia of each segment was recorded and the relative area occupied by dead cells in the exposed epithelium of each segment was calculated using a photograph of the tangentially cut surface of the segment (area, 1.5 × 1.1 mm) by using Adobe Photoshop CS5 Extended (Adobe Systems Inc., San Jose, CA, USA), as described in Son et al (2014) .…”

“…The distributions of dead epithelial cells in P. thunbergii stem cuttings inoculated with B. xylophilus and distilled water were surveyed by Son et al (2014) . Experiments in the present study and that of Son et al (2014) were performed at the same time using the same materials and methods.…”

“…Recent studies have shown that cellulase molecules are localized in the esophageal gland cells of B. xylophilus , secreted from the stylet, and used to enter host cells ( Kikuchi et al, 2004 ; Zhang et al, 2006 ). B. xylophilus is likely to attack one pine cell at a time using the stylet, and migrate for subsequent attacks from the sparse and sporadic distribution of the dead epithelial cells in the cortical resin canals of P. thunbergii ( Son et al, 2014 ). The attacking ability and pattern of B. mucronatus is not yet known.…”

“…In this study, to clarify the contradictions about B. mucronatus migration, the distribution of B. mucronatus in pine stem cuttings was investigated at the tissue level using the fluorescein isothiocyanate-conjugated wheat germ agglutinin (F-WGA) staining technique ( Komatsu et al, 2008 ; Son et al, 2010 ). The attacking pattern of B. mucronatus was also investigated by observing the dead epithelial cells in the cortical resin canals after B. mucronatus inoculation into pine stem cuttings using the Evans blue staining method ( Son et al, 2014 ).…”

Identification of Autophagy in the Pine Wood Nematode Bursaphelenchus xylophilus and the Molecular Characterization and Functional Analysis of Two Novel Autophagy-Related Genes, BxATG1 and BxATG8

“…It has become a severe threat to pine forests worldwide [ 4 , 5 , 6 ]. At present, there are many different hypotheses to explain the pathogenesis of PWD, such as the cellulose (which suggests that the destruction of pine cells is triggered by cell wall-degrading enzymes, such as cellulose), phytotoxin and terpenoid hypotheses [ 7 , 8 , 9 ], but the pathogenic mechanism of B. xylophilus remains unknown.…”