| Pectobacterium sp. cause soft-rot disease on a wide range of plant species. Pectobacterium are diverse and widespread in the environment and previous epidemiological studies have been complicated by difficulty in typing the many atypical isolates recovered. Three DNA-based methods, 16S-23S internal transcribed spacer-PCR/RFLP, multi-locus sequence analysis (MLSA), and pulsed-field gel electrophoresis, were tested to identify the isolates from diseased potato stem and tubers. MLSA generated the most reliable classification for the Pectobacterium isolates. Strains from at least two Pectobacterium clades were present in each field examined and representatives of only three of five Pectobacterium clades were isolated, including P. carotovorum subsp. carotovorum, P. carotovorum subsp. brasiliensis, and P. wasabiae. Hypersensitive response (HR) and DNA hybridization assays revealed that some P. carotovorum and P. wasabiae strains lack the type III secretion system. These T3SS-deficient strains were isolated in multiple fields and in multiple years. Most of the T3SS-deficient strains were similar in virulence on potato stems and tubers to T3SS-encoding strains.;The contribution of the Pectobacterium T3SS to virulence on leaves was explored by constructing P. carotovorum strains deficient in T3SS and the mutant strains were examined in Nicotiana benthamiana. P. carotovorum WPP14 elicited HR-like cell death within 24 hours post-infiltration (hpi) and caused maceration on the edges of this patch of dead plant cells within 48 hpi. The T3SS and DspE were required for the HR-like cell death and disease symptoms. WPP14 was unable to suppress callose formation. Strikingly, within 24 hpi, there was strong callose deposition along leaf veins. Pectobacterium cells were observed along the veins, suggesting that the cells moved within the leaves or grew best along the plant veins. Neither the T3SS nor T2SS was required for the multiplication of WPP14 in leaves. Microarray analysis revealed that the N. benthamiana response to P. carotovorum was essentially the same as the response to Pseudomonas syringae pv. tomato DC3000 T3SS mutant. We were unable to identify N. benthamiana genes differentially expressed due to interaction with the Pectobacterium T3SS, suggesting that the proteins secreted through the Pectobacterium T3SS do not significantly manipulate host gene expression. |
