ASSESSMENT OF THE PROCESS OF MOVEMENT OF XYLELLA FASTIDIOSA WITHIN SUSCEPTIBLE AND RESISTANT GRAPE VARIETIES Project Leader:

We are studying the process of movement of Xylella fastidiosa (Xf) cells between xylem vessels and through plants by analyzing the changing proportion of genetically distinct strains, initially introduced into the plants by distance and time from point of inoculation. We are also determining whether bottlenecks in movement of Xf cells in plants are more extreme in resistant plants than in susceptible plants, and whether this phenomenon can be exploited as a tool to screen germplasm for resistance to Xf. We expect that the process of movement of Xf involves a progressive and sequential colonization of a large number of xylem vessels that is limited by anatomical features of plants (nature of pit membranes and other barriers to vessel to vessel movement in the stem). The resulting in bottlenecks practically limit the number of Xf cells that can move from one vessel to another, and thus constitute a major factor that confers resistance in plants. INTRODUCTION Xylella fastidiosa (Xf) has a rather unique means of colonizing plants and causing symptoms, which make strategies of disease control that are useful in other bacterial diseases ineffective. Many agriculturally important plants besides grapevines, including citrus, almond, alfalfa, and coffee, are susceptible to diseases caused by Xf (Hopkins 1989). The bacterium is transmitted to new host plants during xylem sap feeding by sharpshooter vectors and then multiplies and spreads from the site of inoculation to colonize the xylem; a water transport network of vessels composed of dead, lignified cells. Vessels are interconnected by channels, called bordered pits, that allow the passage of xylem sap but block passage of larger objects due to the presence of a pit membrane (Choat et al. 2003, Esau 1977). Bacterial cells attach to the vessel wall and multiply, forming biofilm-like colonies that can, when sufficiently large, occlude xylem vessels, blocking water transport (Alves et al. 2004, Fry and Milholland 1990a, Newman et al. 2003). In susceptible plants, leaf scorching, fruit shriveling and other symptoms result, likely due to the increased stress of xylem blockage as colonization ensues. However, within the majority of host plants, Xf behaves as a harmless endophyte (Freitag 1951). The population size of Xf in grapevines resistant and susceptible to Pierce’s disease (PD) is highly correlated with symptom expression (Alves et al. 2004, Fry and Milholland 1990a, Fry and Milholland 1990b, Hopkins 1981, Newman et al. 2003, Krivanek and Walker 2004). A much higher proportion of vessels are colonized by Xf in symptomatic tissues that in non-symptomatic tissues (Newman et al. 2003). However we still lack an understanding of the process of colonization and what specifically about high populations of Xf leads to symptom expression. The pathways by which water moves through plants via the xylem are spatially complex. It is simplistic to consider axial water movement in stems via xylem vessels as simple vertical “pipes”. Indeed, xylem vessels themselves often follow complicated paths through a tissue with respect to each other (Figure 1). More importantly, the water in a given vessel is in contact with that of different vessels as well as with those in adjacent tracheids via the many pits in the cell walls (Figure 1). Pits are adjacent to one another on either side of the cell wall and thus come in pairs. The pit membrane is composed of the primary cell wall and middle lamella of adjacent cell walls of the pit pairs (Esau 1977). In a bordered pit the secondary cell wall forms a border over the pit membrane leaving a small opening called a pore. While secondary cell walls can be thickened via the intrusion of lignin and other polymers into the cellulosic matrix of the primary cell wall, pits represent local “thinning” of the primary wall with only a minimal amount of cellulose and pectin, which allows relatively free diffusion of water and solutes from one cell to another. Thus instead of a limited number of vertical “pipes’ that conduct water through a stem, there are thousands of alternative pathways that water might travel in a tissue. The interconnectivity of the xylem cells is presumably one means by which the plant overcomes injuries or other insults that would disrupt the movement of water via a given xylem element by shunting it to adjacent cells. In the context of PD it thus becomes obvious that in order for water movement in a stem to be so restricted that disease develops, a large percentage of the xylem pathways must be blocked for disease to occur. Yet, while over 40% of the xylem vessels in a single section of an infected grape stem may be infested with Xf (Newman et al. 2003) this alone is unlikely to explain water stress. Serial sections of grape tissue however, demonstrated that different xylem vessels are blocked in different cross sections; the percentage of occluded vessels in one of several sections along 5 mm of petiole was five times that of a single cross section. Given that inoculation of grape with Xf must occur in a relatively few sites on a vine, it is clear that the pathogen has the ability to move both axially and radially in xylem tissues. Such extensive movement must take some time, explaining why the disease is “progressive” and appears only several weeks after inoculation.