CHARACTERIZATION OF ERWINIA-CAROTOVORA SUBSPECIES AND DETECTION OF ERWINIA-CAROTOVORA SUBSP ATROSEPTICA IN POTATO PLANTS, SOIL AND WATER EXTRACTS WITH PCR-BASED METHODS

A PCR-RFLP test based on a pectate-lyase encoding gene permits the det ection of several Erwinia carotovora subspecies,but requires complete DNA extraction. This paper reports on the suitability of a simplified PCR-RFLP protocol to characterise E. carotovora strains and on the per formance of PCR, using the same primers, to detect the atroseptica sub species in substrates of epidemiological significance. A collection of 140 strains from various hosts and geographical origins was character ised for biochemical traits and PCR-RFLPs. PCR performed on boiled bac terial suspensions yielded an amplification product of 434 bp in 109 o f the 140 strains. None of the E. carotovora subsp. betavasculorum str ains was amplified, even after complete DNA extraction. RFLPs of the P CR product yielded 24 groups, 3 of which were new. Twenty one groups w ere specific to one subspecies. Several strains biochemically similar to E. carotovora subsp. atroseptica, but growing at 37 degrees C, show ed PCR-RFLP profiles characteristic of E. carotovora subsp. carotovora . Phenetic and cladistic analyses gave three main domains, not strictl y related to hosts or geographical origins. The atroseptica (RFLP grou ps 1 and 2) and wasabiae (group 21) subspecies constituted one of the domains, despite clustering distantly from one another. Host specialis ation and molecular homogeneity suggest a clonal structure within thes e subspecies. Conversely, E. carotovora subsp. odorifera, despite its limited host range and geographical distribution, and E. carotovora su bsp. carotovora showed great molecular diversity, spreading respective ly across five and 19 RFLP groups. These two subspecies shared RFLP gr oups 4, 5 and 6. The tree nodes in the phenograms showed a low robustn ess when bootstrapping the data matrix. PCR coupled with a 48h enrichm ent step in a polypectate-rich medium improved detection thresholds of E. carotovora subsp. atroseptica (1.5.10(2)- 1.5.10(3) bacteria/ml in leaves, stems, and tuber peel extracts to 4.10(7) bacteria/ml in wash water) relative to either immunomagnetic separation coupled with PCR or DAS-ELISA (2.10(5) in plant samples to 2.10(7) bacteria/ml in wash water).