Exploring the Hydraulic Failure Hypothesis of Esca Leaf Symptom Formation

Abstract

Vascular pathogens cause disease in a large spectrum of perennial plants, with leaf scorch being one of the most conspicuous symptoms. Esca in grapevine (Vitis vinifera) is a vascular disease with huge negative effects on grape yield and the wine industry. One prominent hypothesis suggests that vascular disease leaf scorch is caused by fungal pathogen-derived elicitors and toxins. Another hypothesis suggests that leaf scorch is caused by hydraulic failure due to air embolism, the pathogen itself, and/or plant-derived tyloses and gels. In this study, we transplanted mature, naturally infected esca symptomatic vines from the field into pots, allowing us to explore xylem integrity in leaves (i.e. leaf midveins and petioles) using synchrotron-based in vivo x-ray microcomputed tomography and light microscopy. Our results demonstrated that symptomatic leaves are not associated with air embolism. In contrast, symptomatic leaves presented significantly more nonfunctional vessels resulting from the presence of nongaseous embolisms (i.e. tyloses and gels) than control leaves, but there was no significant correlation with disease severity. Using quantitative PCR, we determined that two vascular pathogen species associated with esca necrosis in the trunk were not found in leaves where occlusions were observed. Together, these results demonstrate that symptom development is associated with the disruption of vessel integrity and suggest that symptoms are elicited at a distance from the trunk where fungal infections occur. These findings open new perspectives on esca symptom expression where the hydraulic failure and elicitor/toxin hypotheses are not necessarily mutually exclusive.

Figure 1.

Two-dimensional reconstructions of cross sections from microCT volumes of grapevine leaves. Esca asymptomatic (A and B) and esca symptomatic (C and D) leaf midribs of grapevine plants are shown. After a first scan on intact leaves (A and C), the samples were cut (B and D) just above the scanned area to embolize the vessels and then scanned again. Air-filled (e.g. black arrowheads), water-filled (e.g. white arrowheads), and occluded (e.g. red arrowheads) vessels were counted and their cross-sectional diameters quantified to determine the PLC. The PLC represented by either native embolism (A and C) or occluded vessels (B and D) is given in parentheses. Bars = 100 µm.

Figure 2.

Mean native PLC in midribs and petioles of esca asymptomatic (blue) and esca symptomatic (red) leaves of grapevine plants using microCT imaging. PLC was calculated from the diameter of air-filled vessels in intact leaves, based on the total theoretical hydraulic conductivity of each sample. Error bars represent se, and different letters represent statistically significant differences (least-squares mean differences of fixed effects, P < 0.05; n = sample size).

Figure 3.

Mean occlusion PLC in midribs and petioles of esca asymptomatic (blue) and esca symptomatic (red) leaves of grapevine plants using microCT imaging. PLC was calculated from the diameter of occluded vessels, based on the total theoretical hydraulic conductivity of each sample. Error bars represent se, and different letters represent statistically significant differences (least-squares mean differences of fixed effects, P < 0.05; n = sample size).

Figure 4.

Two-dimensional reconstructions from microCT volumes of esca symptomatic leaves of grapevine. A to C, Iohexol-fed midrib viewed in a longitudinal section (A) and cross sections (B and C). For clarity and orientation, the same three vessels are color coded, and dotted lines represent the locations of the sections relative to each other. The contrasting agent iohexol appears bright white and allows for the identification of the water-transport pathway. The iohexol signal can even be seen in partially occluded vessels (e.g. white arrowhead). Occlusions (i.e. gels or tyloses) can span the entire diameter of the vessel (red arrowheads) or only a portion (yellow arrowheads). After a first scan on intact leaves (A and B), the sample was cut (C) just above the scanned area and scanned again. D, Longitudinal section of a midrib with completely occluded vessels. The presence of occlusions is visible (although obscure) inside the vessel lumen (red arrowheads). E, Longitudinal section of an air-filled midrib (after cutting) with clearly visible occlusions (red arrowheads). F, Frequency distribution of the contact angles between the occlusions and the vessel wall (sample size = 190). Bars = 100 µm.

Figure 5.

Light microscopy images of cross sections of esca symptomatic midribs of grapevine. Cross sections were stained with Toluidine Blue O (A), periodic acid-Schiff reaction (B), Ruthenium Red (C), and Lacmoid Blue (D). Red arrowheads indicate the presence of gels filling entirely the vessel lumen, while black arrowheads indicate the presence of tyloses in vessel lumina. Bars = 100 µm.

Figure 6.

Relationship between the esca symptom severity (expressed as percentage of green tissue per leaf) and the theoretical loss of hydraulic conductivity due to occluded vessels (occlusion PLC) in midribs and petioles of grapevine. Points are grouped by plant: A1 and A2 (blue, asymptomatic) and S1 to S4 (red, symptomatic). The relationship between PLC and green tissue is not significant among symptomatic samples (red points, P = 0.25).