EFFECTS OF CHEMICAL MILIEU ON ATTACHMENT, AGGREGATION, BIOFILM FORMATION, AND VECTOR TRANSMISSION OF XYLELLA FASTIDIOSA STRAINS Project Leader:

We have begun work on the effects of chemical and physical factors, including type of media, pH, media volume, and vessel on the in vitro survival, growth and substrate-attachment of a wild-type and mutant strain of Xylella fastidiosa (Xf). The volume of media in which Xf is incubated appears to override the importance of other variables, including any strain differences. Xf populations incubated in small (200uL) volumes died within 24 h in 50% of assays, but fared better as volumes increased. Preliminary results suggest that attachment to the incubation vessel is greater for wild-type compared to an rpfF mutant that does not produce a cell-cell signaling factor. INTRODUCTION Under natural conditions, Xf attaches to and colonizes the foregut of its leafhopper vectors and the xylem vessels of its plant hosts, creating aggregations of cells attached to their host substrates and surrounded by a polysaccharide matrix, forming a biofilm. Some progress has been made in identifying Xf genes responsible for particular colonizing behaviors, and the use of mutants that disable particular functions (e.g. Newman et al. 2004, Feil et al. 2003) is an invaluable aid to studies of transmission and disease. However, much remains to be learned about what environmental factors (of plant or insect origin) affect colonization; and about how such environmental factors interact with bacterial genetic factors to promote or prevent acquisition, retention and delivery of Xf by the vector. The uptake of Xf cells by the insect and subsequent detachment of Xf as insects probe xylem tissue are essential for vector transmission. These simple requirements, however, belie the more complicated picture that emerges from experimental data. For example, Xf added to xylem sap in artificial diets were taken up but not subsequently transmitted to plants by the vector (Davis et al. 1978, Almeida and Purcell, unpublished). In addition, Xf rpfF mutants, which were unable to produce a cell-cell signaling factor (DSF, diffusible signal factor), were acquired by vectors; but they were not retained and were not transmitted to plants (Newman et al. 2004). Although other studies have shown that Xf could be transmitted within an hour of vector acquisition from plants (Severin 1949, Purcell and Finlay 1979), before anything like a biofilm could form in the foregut, the foregoing data suggest that some rudimentary level of attachment may be necessary for short-term transmission; and that retention, and by implication, colonization and biofilm formation, may be necessary for longer-term ability to transmit. However, the actual role of aggregation/attachment/colonization in the transmission of Xf is still largely unknown. It is clear that both genetic and environmental factors affect colonization of Xf in vitro, as well as in insects and plants. Experiments with site-specific mutants of Xf have yielded insights into the control of aggregation/attachment/colonization phenomena, though not always in completely unambiguous ways. For example, the Xf DSF-deficient mutant formed biofilms and caused severe disease in mechanically inoculated plants, in spite of its inability to colonize the insect foregut (Newman et al. 2004). Cell-cell signaling, therefore, apparently plays different roles in Xf colonization behaviors in insects and plants. In the plant pathogen Xanthamonas campestris, DSF triggered dispersion of cell aggregates in vitro, and was suggested to promote virulence to plants (Dow et al. 2003). Mutants in two other Xf genes involved in formation of bacterial fimbriae that aid in attachment, fimA and fimF, showed reduced aggregation in vitro, but were insect transmissible, and caused disease in grapevines (Feil et al. 2003, Feil and Purcell, unpublished). In both the plant and the vector, environmental factors that putatively affect attachment or detachment would include chemical makeup of sap from which Xf cells are acquired; the substrate colonized (insect foregut, xylem vessels); and movement of sap through the xylem or foregut. Media composition has a reportedly major effect on aggregation and biofilm formation of Xf (Leite et al. 2004). It is likely that substrate surface characteristics are also important, by analogy with