Citrus sinensis leaf petiole and blade colonization by Xylella fastidiosa: details of xylem vessel occlusion

Eduardo Alves,Peter Craig Andersen,Maria Lúcia Ishida,Breno Leite,Sérgio Florentino Pascholati

doi:10.1590/s0103-90162009000200011

Abstract

Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is an important disease of citrus in Brazil. X. fastidiosa is restricted to xylem vessels of plants and knowledge regarding xylem colonization is still limited. Our goal was to verify how this bacterium colonizes and spreads within xylem vessels of sweet orange Citrus sinensis cv. Pêra. Petioles and pieces of leaf blades from naturally infected plant exhibiting characteristic symptoms were prepared for light microscopy (LM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and immunogold labeling (IGL). Petioles from healthy plants were used as control. IGL results, using an antibody against wall hemicelluloses, revealed that the pit membrane of vessels was altered. Bacterial cells were observed in the pit between adjacent vessels. Results support the contention that X. fastidiosa produces cellulases to reach adjacent vessels. SEM revealed that colonization of sweet orange started with X. fastidiosa cells attaching to the xylem wall, followed by an increase in the number of bacterial cells, the production of fibrous material, and finally vessel occlusion by biofilm composed of copious amounts of amorphous material, strands and cells. Phenolic materials, hyperplasia and hypertrophy were noticed in leaves with gummy material. Xylem vessels frequently contained an unknown needle-like, crystallized matter blocking the vessel.

Highlights

Xylella fastidosa (Wells et al, 1987) is a xylem-limited bacterium that has affected many economically-important crops worldwide (Purcell, 1997)
scanning electron microscopy (SEM) revealed that colonization of sweet orange started with X. fastidiosa cells attaching to the xylem wall, followed by an increase in the number of bacterial cells, the production of fibrous material, and vessel occlusion by biofilm composed of copious amounts of amorphous material, strands and cells
The observations suggest that the colonization involve the initial invasion of pitted vessels (Figures 1C and 1D). These vessels are separated from neighbor vessels by the pit membrane within the pits, which consists of a middle lamella and a primary cell wall

Summary

Introduction

Xylella fastidosa (Wells et al, 1987) is a xylem-limited bacterium that has affected many economically-important crops worldwide (Purcell, 1997). Stem dieback and reduction of fruit size are observed (Feichtenberger et al, 2005) These symptoms are a result of vessel obstruction due to systemic colonization of bacteria (Sherald & Lei, 1991) and the production of fastidian gum within the xylem vessel lumen (Silva et al, 2001). Xylem fluid chemistry is under investigation for its possible role to induce cell aggregation and biofilm formation (Leite et al, 2004b; Andersen, et al, 2005). Hopkins (1989) suggested that X. fastidiosa cells could degrade the pit membrane between two xylem vessels to promote intervessel migration. Precursor genes of the polygalacturonase and celullase were identified by the bacterial genome analysis (Simpson et al, 2000) suggesting that X. fastidiosa is capable of degrading pit membranes. Wuff et al (2003) cloned and expressed X. fastidiosa cellulase genes

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