| Xylella fastidiosa (Xf) is a xylem-limited, nutritionally fastidious bacterial plant pathogen that causes Pierce's disease (PD) in grape, citrus variegated chlorosis (CVC), almond leaf scorch (ALS), oleander leaf scorch (OLS), as well as diseases of several other trees and ornamentals. A PCR-based method was developed to distinguish among strains of the subspecies fastidiosa, sandyi and multiplex. Primers, XF1968-L and XF1968-R amplified a 638 by PCR-fragment from sandyi subspecies but not from fastidiosa subspecies, whereas primers XF2542-L and XF2542-R amplified a 412 by PCR fragment from fastidiosa subspecies but not from sandyi subspecies. Furthermore, primers ALM1 and ALM2 produced a fragment of 521 bp from strains isolated from almond that belong to multiplex subspecies. The combination of the three primer sets allowed the distinction of two groups of ALS strains within the multiplex subspecies. Strains isolated from ornamentals were characterized using a multiprimer PCR system, RAPD-PCR, and sequence analysis of the 16S-23S rDNA intergenic spacer regions. The results of the combined methods revealed new hosts for the subspecies fastidiosa (peach and redbud) multiplex (crape myrtle, gingko, olive, liquidambar, purple-leaf plum and redbud), sandyi (daylily, magnolia and jacaranda), and mulberry leaf scorch (heavenly bamboo). It was also shown that strains isolated from jacaranda, daylily, and magnolia are able to produce disease in oleander but not in grape. Finally, to identify genes involved in pathogenicity, the genome sequence of the CVC strain 9a5c was used to select open reading frames (ORFs) with similarity to pathogenicity and virulence factors in other bacterial species. Primer pairs were designed for 111 ORFs, amplified and arrayed onto nylon membranes. These arrays were hybridized with 32P-labeled cDNA prepared from different strains of Xylella cultured in vitro and from infected purple-leaf plum and oleander plants. Macroarray analysis of six different strains in PD3 media revealed that the expression levels for those genes were not significantly different between strains. In contrast, six genes were found to be differentially expressed in infected plants: higB, acvB, hsf, b0819, rpfA and surE. In each case, results were verified by RT-PCR. |
