, Volume 104, Issue 7, pp 685-699

Characterisation of Erwinia carotovora subspecies and detection of Erwinia carotovora subsp. atroseptica in potato plants, soil and water extracts with PCR-based methods

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Abstract

A PCR-RFLP test based on a pectate-lyase encoding gene permits the detection 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 performance of PCR, using the same primers, to detect the atroseptica subspecies in substrates of epidemiological significance. A collection of 140 strains from various hosts and geographical origins was characterised for biochemical traits and PCR-RFLPs. PCR performed on boiled bacterial suspensions yielded an amplification product of 434 bp in 109 of the 140 strains. None of the E. carotovora subsp. betavasculorum strains was amplified, even after complete DNA extraction. RFLPs of the PCR product yielded 24 groups, 3 of which were new. Twenty one groups were specific to one subspecies. Several strains biochemically similar to E. carotovora subsp. atroseptica, but growing at 37 °C, showed PCR-RFLP profiles characteristic of E. carotovora subsp. carotovora. Phenetic and cladistic analyses gave three main domains, not strictly related to hosts or geographical origins. The atroseptica (RFLP groups 1 and 2) and wasabiae (group 21) subspecies constituted one of the domains, despite clustering distantly from one another. Host specialisation and molecular homogeneity suggest a clonal structure within these subspecies. Conversely, E. carotovora subsp. odorifera, despite its limited host range and geographical distribution, and E. carotovora subsp. carotovora showed great molecular diversity, spreading respectively across five and 19 RFLP groups. These two subspecies shared RFLP groups 4, 5 and 6. The tree nodes in the phenograms showed a low robustness when bootstrapping the data matrix. PCR coupled with a 48h enrichment step in a polypectate-rich medium improved detection thresholds of E. carotovora subsp. atroseptica (1.5.102- 1.5.103 bacteria/ml in leaves, stems, and tuber peel extracts to 4.107 bacteria/ml in wash water) relative to either immunomagnetic separation coupled with PCR or DAS-ELISA (2.105 in plant samples to 2.107 bacteria/ml in wash water).