Applying volumetric electron microscopy to visualize xylem tissue impacted by citrus tristeza virus-induced stem pitting

Abstract

Citrus tristeza virus (CTV) causes several disease syndromes in different citrus hosts: quick decline, seedling yellows and stem pitting. CTV-induced stem pitting leads to substantial economic losses in sensitive citrus varieties, including grapefruit. The formation of stem pits has previously been linked to the ability of the virus to colonize xylem tissue outside of its typical phloem limitation, thereby disrupting normal xylem development. The nature of this compromised tissue has not been fully elucidated. In this study, stem pits were characterized at the molecular anatomical level using a combination of techniques to better understand the characteristics of the xylem and phloem tissues impacted by severe pitting. Biological staining was used to visualize CTV-induced stem pitting and was complemented with a novel technology that has not previously been used to study CTV-induced stem pitting, namely serial block-face scanning electron microscopy (SBF-SEM). This proof-of-concept study yielded new insights into the morphology of stem pitting-affected tissue. The utility of SBF-SEM for stem pitting characterization was also demonstrated and an optimized protocol for its application on hard, woody material is presented.