The structure of xylem vessels in grapevine (Vitaceae) and a possible passive mechanism for the systemic spread of bacterial disease

Xylem-dwelling pathogens become systemic, suggesting that microorganisms move efficiently in the xylem. To better understand xylem pathways and how bacteria move within the xylem, vessel connectivity between stems and leaves of Vitis vinifera cv. Chardonnay and Muscadinia rotundifolia cv. Cowart was studied. Three methods were used: (1) the light-producing bacterium, Yersinia enterocolitica, (Ye) strain GY5232 was loaded into petioles and followed using X-ray film, (2) fluorescent beads were loaded and followed by microscopy, and (3) low-pressure air was pumped into leaves and extruded bubbles from cuts in submerged leaves were followed. Bacteria, beads, and air moved through long and branched xylem vessels from the petiole into the veins in leaves of both varieties. From the stem, bacteria and air traveled into primary and secondary veins of leaves one, two, and three nodes above the loading point of the bacteria or air. Particles and air could move unimpeded through single xylem vessels or multiple vessels (conduits) connected possibly through broken pit membranes from within the stem axis into leaf blades. Bacteria were also able to move long distances within minutes from stem to leaf passively without having to cross pit membranes. Such complex, open xylem conduits have not been well documented before; these findings will help elucidate mechanisms involved in the systemic spread of pathogens.     

Xylem structure of four grape varieties and 12 alternative hosts to the xylem-limited bacterium Xylella fastidious

Background and Aims

The bacterium Xylella fastidiosa (Xf), responsible for Pierce''s disease (PD) of grapevine, colonizes the xylem conduits of vines, ultimately killing the plant. However, Vitis vinifera grapevine varieties differ in their susceptibility to Xf and numerous other plant species tolerate Xf populations without showing symptoms. The aim of this study was to examine the xylem structure of grapevines with different susceptibilities to Xf infection, as well as the xylem structure of non-grape plant species that support or limit movement of Xf to determine if anatomical differences might explain some of the differences in susceptibility to Xf.

Methods

Air and paint were introduced into leaves and stems to examine the connectivity between stem and leaves and the length distribution of their vessels. Leaf petiole and stem anatomies were studied to determine the basis for the free or restricted movement of Xf into the plant.

Key Results

There were no obvious differences in stem or petiole vascular anatomy among the grape varieties examined, nor among the other plant species that would explain differences in resistance to Xf. Among grape varieties, the more tolerant ‘Sylvaner’ had smaller stem vessel diameters and 20 % more parenchyma rays than the other three varieties. Alternative hosts supporting Xf movement had slightly longer open xylem conduits within leaves, and more connection between stem and leaves, when compared with alternative hosts that limit Xf movement.

Conclusions

Stem–leaf connectivity via open xylem conduits and vessel length is not responsible for differences in PD tolerance among grape varieties, or for limiting bacterial movement in the tolerant plant species. However, it was found that tolerant host plants had narrower vessels and more parenchyma rays, possibly restricting bacterial movement at the level of the vessels. The implications of xylem structure and connectivity for the means and regulation of bacterial movement are discussed.     

Xylella fastidiosa infection and ethylene exposure result in xylem and water movement disruption in grapevine shoots

It is conventionally thought that multiplication of the xylem-limited bacterium Xylella fastidiosa (Xf) within xylem vessels is the sole factor responsible for the blockage of water movement in grapevines (Vitis vinifera) affected by Pierce's disease. However, results from our studies have provided substantial support for the idea that vessel obstructions, and likely other aspects of the Pierce's disease syndrome, result from the grapevine's active responses to the presence of Xf, rather than to the direct action of the bacterium. The use of magnetic resonance imaging (MRI) to observe the distribution of water within the xylem has allowed us to follow nondestructively the development of vascular system obstructions subsequent to inoculation of grapevines with Xf. Because we have hypothesized a role for ethylene produced in vines following infection, the impact of vine ethylene exposure on obstruction development was also followed using MRI. In both infected and ethylene-exposed plants, MRI shows that an important proportion of the xylem vessels become progressively air embolized after the treatments. The loss of xylem water-transporting function, assessed by MRI, has been also correlated with a decrease in stem-specific hydraulic conductivity (K(S)) and the presence of tyloses in the lumens of obstructed water conduits. We have observed that the ethylene production of leaves from infected grapevines is greater than that from healthy vines and, therefore, propose that ethylene may be involved in a series of cellular events that coordinates the vine's response to the pathogen.     

The effects of Pierce's disease on leaf and petiole hydraulic conductance in Vitis vinifera cv. Chardonnay

In this study, we test the hypothesis that the symptoms of Pierce's Disease (PD) result from the occlusion of xylem conduits by the bacteria Xylella fastidiosa ( Xf ). Four treatments were imposed on greenhouse-grown Vitis vinifera cv. Chardonnay: well-watered and deficit-irrigated plants with and without petiole inoculation with Xf . The hydraulic conductance of the stem-petiole junction ( k _jun) and leaves ( k _leaf) were measured, and Xf concentrations were established by quantitative polymerase chain reaction (qPCR). Leaf hydraulic conductance decreased with increasing leaf scorch symptoms in both irrigation treatments. The positive relationship between Xf concentration and symptom formation in deficit-irrigated plants suggests that water-stress increases susceptibility to PD. In field-grown vines, water relations of symptomatic leaves were similar to naturally senescing leaves but differed from green control leaves. Overall, these results suggest that the development of PD symptoms represents a form of accelerated senescence as part of a systemic response of the plant to Xf infection.     

Leaf scorch symptoms are not correlated with bacterial populations during Pierce's disease

Xylella fastidiosa (Xf) is a xylem-limited bacterium that lives as a harmless endophyte in most plant species but is pathogenic in several agriculturally important crops such as coffee, citrus, and grapevine (Vitis vinifera L.). In susceptible cultivars of grapevine, Xf infection results in leaf scorch, premature leaf senescence, and eventually vine death; a suite of symptoms collectively referred to as Pierce's disease. A qPCR assay was developed to determine bacterial concentrations in planta and these concentrations were related to the development of leaf-scorch symptoms. The concentration of Xf in leaves of experimental grapevines grown in the greenhouse was similar to the concentration of Xf in leaves of naturally infected plants in the field. The distribution of Xf was patchy within and among leaves. Some whole leaves exhibited severe leaf-scorch symptoms in the absence of high concentrations of Xf. Despite a highly sensitive assay and a range of Xf concentrations from 10(2) to 10(9) cells g(-1) fresh weight, no clear relationship between bacterial population and symptom development during Pierce's disease was revealed. Thus, high and localized concentrations of Xf are not necessary for the formation of leaf-scorch symptoms. The results are interpreted as being consistent with an atiology that involves a systemic plant response.     

Co-ordinated development of the leaf midrib xylem with the lamina in Nicotiana tabacum

Background and Aims

The water-transport capacity of leaf venation is positively related to the leaf-lamina area, because the number and diameter of vein-xylem conduits are controlled to match the lamina area. This study aimed to investigate how this co-ordinated relationship between the leaf-lamina area and vein-xylem characteristics is achieved by examining the midrib xylem of tobacco leaves.

Methods

The changes in the midrib-xylem characteristics over time were quantified using leaves with four different final lamina areas. The measured data were fitted to sigmoidal functions. From the constants of the fitted curves, the final values in mature leaves, maximal developmental rates (V_Dev) and developmental duration (T_Dev) were estimated for each of the xylem characteristics. Whether it is the lamina or the midrib xylem that drives the co-ordinated development was examined by lamina removal from unfolding leaves. The effects of the application of 0·1 % IAA (indole-3-acetic acid) to leaves with the laminas removed were also analysed.

Key Results

For both the leaf lamina and the midrib-xylem characteristics, the differences in final values among leaves with different lamina areas were more strongly associated with those in V_Dev. Notably, the V_Dev values of the midrib-xylem characteristics were related to those of the leaf-lamina area. By lamina removal, the conduit diameter was reduced but the number of conduits did not significantly change. By IAA application, the decrease in the conduit diameter was halted, and the number of conduits in the midrib xylem increased.

Conclusions

According to the results, the V_Dev values of the lamina area and the midrib-xylem characteristics changed in a co-ordinated manner, so that the water-transport capacity of the midrib xylem was positively related to the leaf-lamina area. The results also suggest that IAA derived from the leaf lamina plays a crucial role in the development of the leaf venation.     

Hydraulic disruption and passive migration by a bacterial pathogen in oak tree xylem

Xylella fastidiosa (Xf) is a xylem-limited bacterial pathogen that causes leaf scorch symptoms in numerous plant species in urban, agricultural, and natural ecosystems worldwide. The exact mechanism of hydraulic disruption and systemic colonization of xylem by Xf remains elusive across all host plants. To understand both processes better, the functional and structural characteristics of xylem in different organs of both healthy and Xf-infected trees of several Quercus species were studied. Hydraulic conductivity (K(s)) in Xf-infected petioles of Q. palustris and Q. rubra decreased significantly compared with healthy trees as the season progressed and plummeted to zero with the onset of scorch symptoms. Prior to the onset of symptoms, embolism was as much as 3.7 times higher in Xf-infected petioles compared with healthy controls and preceded significant reductions in K(s). Embolism likely resulted from pit membrane degradation during colonization of new petiole xylem and triggered the process that eventually led to vessel occlusion. Pit membrane porosity was studied using the following four methods to determine if a pathway exists in the xylem network of woody stems that allows for passive Xf migration: (i) calculations based on vulnerability to cavitation data, (ii) scanning electron micrographs, (iii) microsphere injections, and (iv) air seeding thresholds on individual vessels. All four methods consistently demonstrated that large pit membrane pores (i.e. greater than the diameter of individual Xf) occur frequently throughout the secondary stem xylem in several Quercus species. These large pores probably facilitate systemic colonization of the secondary xylem network and contribute to the high susceptibility to bacterial leaf scorch exhibited among these species.     

The spatial pattern of air seeding thresholds in mature sugar maple trees

Air seeding threshold (P_a) of xylem vessels from current year growth rings were measured along the vertical axis of mature sugar maple trees (Acer saccharum Marsh.), with sampling points in primary leaf veins, petioles, 1-, 3-, and 7-year-old branches, large branches, the trunk and roots. The air seeding threshold was taken as the pressure required to force nitrogen gas through intervessel pit membranes. Although all measurements were made on wood produced in the same year, P_a varied between different regions of A. saccharum, with distal organs such as leaves and petioles having lower P_a than basal regions. Mean (SE) P_a ranged from 1.0 (± 0.1) MPa in primary leaf veins to 4.8 (± 0.1) MPa in the main trunk. Roots exhibited a P_a of 2.8 (± 0.2) MPa, lower than all other regions of the tree except leaf veins and petioles. Mean xylem vessel diameter increased basipetally, with the widest vessels occurring in the trunk and roots. Within the shoot, wider vessels had greater air seeding thresholds, contrasting with trends previously reported. However, further experimentation revealed that differences in P_a between regions of the stem were driven by the presence of primary xylem conduits, rather than differences in vessel diameter. In 1-year-old branches, P_a was significantly lower in primary xylem vessels than in adjacent secondary xylem vessels. This explained the lower values of P_a measured in petioles and leaf veins, which possessed a greater ratio of primary xylem to secondary xylem than other regions. The difference in P_a between primary and secondary xylem was attributed to the greater area of primary cell wall (pit membrane) exposed in primary xylem conduits with helical or annular thickening.     

The Development of the Runner-bean Leaf with Special Reference to the Relation between the Sizes of the Lamina and of the Petiolar Xylem: I. The Relation between Lamina Area and Petiolar Xylem

An investigation has been made of the relation between the sizesof the lamina and of the petiolar xylem of both mature and immatureleaves of the runner bean (Phaseolus multiflorus Willd.). The ratios xylem area/lamina area and the number of vessels/laminaarea are lowest for mature leaves. Immature leaves gave higherbut somewhat more variable values for these two ratios. There is a constant growth ratio between the lpminn area andthe xylem area, such that k is approximately o·61 inthe allometry formula. The significance of the results is briefly considered from thepoint of view that the xylem area is related to the water requirementsof the leaf.     

The Development of the Runner-bean Leaf with Special Reference to the Relation between the Sizes of the Lamina and of the Petiolar Xylem: II. The Normal Development of the Bean Leaf

The development of the first pair of leaves of the Runner-bean(Phaseolus multiflorus Willd.) has been investigated from theunfolding of the leaves to the end of lamina expansion. Thegrowth of the petiole is relatively fast and ceases first. Extensiongrowth of the lamina continues longer. In the growth of thexylem, the average area of the vessels formed increases mostmarkedly at an early developmental stage. Vessel number maycontinue to increase after lamina growth has ceased. Both thenumber of vessels and the xylem area per unit area of laminadiminish as the leaf matures.     

An analysis of resistance to water flow through wheat and tall fescue leaves during pressure chamber efflux experiments

Abstract. This is a physical analysis of water movement in wheat ( Triticum ) and tall fescue ( Festuca arundinacea ) leaves placed in the Scholander pressure chamber. It takes into account the efflux resistances of water movement through the xylem and water flow across the cell membranes. Xylem resistance was estimated using Poiseuille's law.
Leaves which had been pressurized in the chamber were embedded, sectioned, examined under a light microscope and photographed. Cells were intact but distorted and xylem vessels were intact. Cells in portions of the blade squeezed by the chamber sealing grommet were crushed, but xylem vessels remained intact.
By applying pressure several tenths of a megapascal in excess of the balance pressure, water was forced from each leaf through the severed end which protruded from the chamber. Efflux curves were drawn by plotting the total water expressed as a function of time after the pressure increase. Water efflux from the shortest wheat leaf lasted only 10 min while efflux from the longest continued for up to 40 min. The efflux from a tall fescue leaf which was rehydrated and cut to a shorter length was much more rapid than efflux from the original leaf.
Experiments combined with mathematical analysis suggested that the effect of leaf length on efflux is related to a high resistance to water flow through vascular bundles. Xylem resistance would be sufficient to produce this effect if it were 10 times greater than that predicted by Poiseuille's law. Both the observations of water flow from the cut end of the leaf and the mathematical model suggested very little water flows from bundles with vessels of diameter less than 12 μm. The apparent explanation is high resistance to water flow through these small diameter vessels.     

THE PRODUCTION AND DIFFERENTIATION OF SECONDARY XYLEM IN XANTHIUM PENSYLVANICUM

The removal of leaves and buds from the shoot of Xanthium seedlings caused the cessation of xylem fiber differentiation in all internodes while allowing the production of cambial derivatives to continue toward both the xylem and the phloem. The potential xylem fibers developed into parenchymatous cells with thin cell walls. The vessels developed normally except for their small size. Cambial derivatives and vessels were produced linearly with time in intact plants (6.1 cells per file/day and 9.7 vessels per vascular bundle/day) and in decapitated plants (2.2 cells per file/day and 5.5 vessels per vascular bundle/day). Fiber production was linear with time in intact plants (163 fibers per vascular bundle/day) and did not occur in decapitated plants. When a single leaf was allowed to develop from a lateral bud of a decapitated plant, xylem fiber differentiation was restored for a period of time corresponding to the period of rapid expansion of the leaf blade. When the leaf passed the phase of rapid expansion, it no longer had an inductive effect on xylem fiber differentiation.     

Changes in stomatal conductance along grass blades reflect changes in leaf structure

Identifying the consequences of grass blade morphology (long, narrow leaves) on the heterogeneity of gas exchange is fundamental to an understanding of the physiology of this growth form. We examined acropetal changes in anatomy, hydraulic conductivity and rates of gas exchange in five grass species (including C(3) and C(4) functional types). Both stomatal conductance and photosynthesis increased along all grass blades despite constant light availability. Hydraulic efficiency within the xylem remained constant along the leaf, but structural changes outside the xylem changed in concert with stomatal conductance. Stomatal density and stomatal pore index remained constant along grass blades but interveinal distance decreased acropetally resulting in a decreased path length for water movement from vascular bundle to stomate. The increase in stomatal conductance was correlated with the decreased path length through the leaf mesophyll. A strong correlation between the distance from vascular bundles to stomatal pores and stomatal conductance has been identified across species; our results suggest this relationship also exists within individual leaves.     

The flow and dispersion of water in the vascular network of dicotyledonous leaves

The relationships between the geometric characteristics of, the local flow rates of xylem sap in, and relative pressures in the reticulate anastomosing vascular network of dicotyledonous leaves of Populus Balsamifera L. are reported. The conducting channels of cellulosic microcapillaries are covered by sheaths of chloroplast free cells through the walls of which water withdrawn from vascular bundles percolates to reach evaporation sites. Along the mid-rib and branch generations, the population and cross-section areas of the microcapillaries decrease with distance but not in a monotonic manner. Lateral withdrawal rates from the veins were highest at the base of the leaf lamina. More than 50% of the inlet stream had dispersed out of the conduits within the first 25% of the leaf lamina area from the petiole junction. Absolute values of pressure gradients generally decreased in the apical direction along the vein.     

Changes in Stem and Leaf Hydraulics Preceding Leaf Shedding in Castanea Sativa L.

This paper describes changes in leaf water status and in stem, petiole and leaf blade hydraulics preceding leaf senescence and shedding in Castanea sativa L. (chestnut). Measurements of maximum diurnal leaf conductance to water vapour (g_L), minimum water potential (_L), hydraulic conductance per unit leaf surface area of stems (K_SL), petioles (K_PL) and leaf blades (K_LL) and number of functional conduits and inside diameter distribution were performed in June, September and October 1999. In September, still green leaves had undergone some dehydration as indicated by decreased g_L (by 75 %) and _L with respect to June. In the same time, K_SL decreased by 88 %, while K_PL and K_LL decreased by 50 % and 20 % of the conduits of stems and 10 % of the petioles (all belonging to the widest diameter range) were no longer functioning, causing a decrease in the theoretical flow by 82 % in stems and 27 % in petioles. Stem xylem blockage was apparently due to tyloses growing into conduits. We advance the hypothesis that the entire process of leaf shedding and winter rest may be initiated by extensive stem embolism occurring during the summer.     

A wilty mutant of rice has impaired hydraulic conductance

The rice CM2088 mutant is the wilty phenotype and wilts markedly under well-watered sunny conditions. The leaf water potential and epidermal (mainly stomatal) conductance of CM2088 plants decreased significantly under conditions that induced intense transpiration, as compared with those of wild-type plants, revealing that the wilty phenotype was not the result of abnormal stomatal behavior but was due to an increase in resistance to water transport. The resistance to water transport was dramatically elevated in the node and the sheath and blade of a leaf of the mutant, but not in the root or stem. The diameter of xylem vessels in the large vascular bundles of the leaf sheath and the internode tended to be small, and the numbers of vessel elements with narrowed or scalariform perforation plates in the leaf blade and sheath were greater in the mutant than in the wild type. Most xylem vessels were occluded, with air bubbles in the leaf sheath of the mutant during the midday hours under intense transpiration conditions, while no bubbles were observed in plants that were barely transpiring, revealing that the significant increase in resistance to water transport was a result of the cavitation. The additive effects of cavitation in xylem vessels and the decreased diameter and deformed plates of vessel elements might be responsible for the wilty phenotype of CM2088.     

The relation of anatomy to water movement and cellular response in young barley leaves

Young barley (Hordeum vulgare L. cv Arivat) leaves were examined anatomically and physiologically to infer the pathway of transpirational water movement and to explain why the growing region is more responsive to osmotic stress than the expanded blade. Vessels with open lumens extend from the intercalary meristem to the expanded blade, and all vessels are clustered in five vascular bundles that are separated by 20 closely packed mesophyll cells. Heat pulse transport data confirmed the anatomical suggestion that water moves through the growing region in vessels and not intercellularly, and also showed that stress reduces xylem water transport within 1 minute while transpiration remained unaffected. Water equal in volume to twice that expected in the xylem, and which exchanges more readily with water in the nutrient solution than with most water in tissues, can be extracted easily from growing tissues. It is hypothesized that this water is xylem plus cell wall water, that osmotic stress will quickly reduce its in situ water potential, and that stress causes growth to stop because cells in the growing region can respond rapidly to changes in water potential around them. In the expanded blade, bundles containing vessels are three and eight cells away from the closest and next substomatal cavities. This allows xylem water loss to occur predominantly through the closest stomata, and the expanded blade is believed to be less responsive because effects of stress on xylem water potential are confined largely to cells immediately around the vessels.     

Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera

Xylella fastidiosa causes Pierce's disease of grapevine as well as several other major agricultural diseases but is a benign endophyte in most host plants. X. fastidiosa colonizes the xylem vessels of host plants and is transmitted by xylem sap-feeding insect vectors. To understand better the pattern of host colonization and its relationship to disease, we engineered X. fastidiosa to express a green fluorescent protein (Gfp) constitutively and performed confocal laser-scanning microscopic analysis of colonization in a susceptible host, Vitis vinifera. In symptomatic leaves, the fraction of vessels colonized by X. fastidiosa was fivefold higher than in nearby asymptomatic leaves. The fraction of vessels completely blocked by X. fastidiosa colonies increased 40-fold in symptomatic leaves and was the feature of colonization most dramatically linked to symptoms. Therefore, the extent of vessel blockage by bacterial colonization is highly likely to be a crucial variable in symptom expression. Intriguingly, a high proportion (>80%) of colonized vessels were not blocked in infected leaves and instead had small colonies or solitary cells, suggesting that vessel blockage is not a colonization strategy employed by the pathogen but, rather, a by-product of endophytic colonization. We present evidence for X. fastidiosa movement through bordered pits to neighboring vessels and propose that vessel-to-vessel movement is a key colonization strategy whose failure results in vessel plugging and disease.     

Ultrasound acoustic emissions from dehydrating leaves of deciduous and evergreen trees

While drying, detached leaves produced ultrasound acoustic emissions (UAE) comparable to emissions from stem and twig wood. Experiments on Ilex aquifolium L. showed that the main source of these signals was cavitation in the veins, to which conduits and fibres probably both contributed. Regions of the leaf blade with abundant mesophyll and only small veins emitted few signals. More signals were counted on the adaxial side of the midrib than on the abaxial one and on the proximal third than on the distal one, in accordance with the anatomical structure. Sound attenuation was pronounced. Eight species were compared with respect to cavitation behaviour, field water relations and pressure–volume curves, and the data showed differences in cumulative number of events and resistance of leaves to cavitation. Data were generally in good agreement with anatomical structure and habitat preferences. The number of signals per conduit counted on cross-sections was in some leaves much higher than unity, which suggests short xylem elements or an acoustic activity of cells other than conduits. There was no correlation between cavitation threshold or cumulative number of signals and the degree of sclerophylly; unexpectedly, there was a correlation between the cumulative number of signals at a water potential of -1.3 MPa and the bulk modulus of elasticity.     

The Development of the Runner Bean Leaf with Special Reference to the Relation between the Sizes of the Lamina and of the Petiolar Xylem: III. The Development of the Leaf under Various Conditions

The development of the first pair of leaves of the Runner Bean(Phaseolus multiflorus Willd.) under various conditions is described. 1. Under damp conditions and also when one leaf of a pair wascovered with a black paper envelope, the rate of developmentwas altered but no significant changes in the relative developmentof the parts of the leaf occurred. 2. Removal of a portion of the lamina at an early stage of developmentresulted, at maturity, in a leaf having relatively less petiolarxylem, although the ratio xylem area/lamina area was slightlyhigher. This treatment also delayed a ‘sudden’ increasein area of the vessels which was seen in normal plants. 3. An attempt was made to estimate the transporting power ofthe petiolar xylem and to compare it with the lamina area. Thedata suggest that the maximum possible area of the lamina atany stage in development may be determined by the transportingpower of the xylem, but that the attainment of this maximumpossible area may be prevented by external factors as is seenin some of the experimental results presented.