BIOLOGICAL, CULTURAL, AND CHEMICAL MANAGEMENT OF PIERCES DISEASE

• Author(s): Andersen, Peter; Kirkpatrick, Bruce; Purcell, Alexander; Walker, Andrew; Weber, Ed;
• Abstract: The systemic movement of Xylella fastidiosa (Xf) within the plant xylem system is essential to this bacteriums ability to cause disease and probably for its indefinite survival in natural environments. Numerous microscopic studies of plants affected by Pierces disease revealed high concentrations of bacteria in some xylem cells, but it is notable that in all of these studies, adjacent xylem elements are often devoid of bacteria. The important basic question of how the bacteria move from cell to cell is still unanswered. Our initial hypothesis is that bacterial multiplication is an important requisite for cell-to-cell movement. Do Xf populations in nonsystemic hosts reach high or low densities within infected cells? Do Xf populations in systemic hosts with low populations of Xf (as determined by dilution plating) attain high populations in few cells or lower populations in many cells? We will examine the behavior of Xf in nonsystemic hosts such as willow and mugwort and in plants with low, but systemic populations, such as blackberry. The occlusion of xylem cells with Xf in willow, for example, would illustrate that bacterial aggregates that completely fill xylem cells is not sufficient for systemic movement. The Xf oleander strain is not systemic in grape and vice versa (A.H. Purcell et al., unpublished data). The typical fate of Xf in most woody plant species is to multiply without systemic movement (A.H. Purcell, unpublished). We will investigate if this is true for the oleander strain in grape and the grape strain in oleander. Both of these strains are systemic in their pathological hosts but not in the opposite host. We will seek to identify whether oleander strains multiply in grape using dilution plating on solid culture medium (PW) and confocal fluorescent microscopy. A recent development that aids the study of bacterial movements in plants is the emergence of scanning confocal laser microscopy (SCLM). The new techniques to study bacterial biofilms could provide valuable information on the distribution of Xf within xylem tissue. The application of the SCLM coupled with image analysis techniques permits the study of living, fully hydrated microbial biofilms. Success in introducing novel genes into Xf (objective 4) to create new but grape-virulent strains with reporter gene constructs allowing easy Xf detection in grape tissues would greatly facilitate studies of Xf movement in plants. Such a system could enable Xf detection with SCLM in plants without fixing, dehydrating, staining, or otherwise preparing plant specimens. Thus the same tissues could be examined repeatedly to follow Xf movements, especially those events associated with the cell to cell movements that are critical to disease. If Xf can be genetically engineered to express the green fluorescent protein (GFP) gene, the movement of bacteria could be followed through the plant similar to the methods used in studies on the movement of Erwinia amylovora in apple. Our use of GFP-mutants for histological studies of movements or of biofilm formation will depend upon success of objective 4. An understanding of biofilms may also help explain Xf movement and pathogenicity. A biofilm is an aggregate of attached cells produced when bacteria adhere to a surface, initiate glycocalyx (exopolysaccharide) production and form microcolonies. Xf appears to produce biofilms that are unique compared to other documented biofilms in that they are inhabited only by a single bacterial species and occur within plant xylem and insect guts. Others have speculated the matrix material surrounding aggregations of Xf within plants improves the bacteriums extraction of nutrients and provide physical protection. We suspect that biofilm formation is also critical to Xfs movement from cell to cell within plants and necessary for its survival within the vector foregut, from which it is transmitted to plants by insects. We examine Xfs occurrence within a spectrum of host plants - from those in which it multiplies rapidly but does not exhibit systemic spread (willow), to those in which it multiplies and moves but does not reach high enough levels to cause disease, to pathological hosts such as grape. We will also develop methods to produce and examine biofilms under in vitro conditions so as to be able to experimentally manipulate environmental conditions and determine their effects on biofilm formation. If we are successful in developing Xf transformation protocols (objective 4), we should be able to provide conclusive genetic evidence for the potential role of biofilms in plant pathogenesis or insect transmission by knocking out biofilm biosynthesis gene(s) using transposon mutagenesis.
• Publication Date: Dec 2001
• Journal: 2001 Pierce's Disease Research Symposium