Pectobacterium parmentieri causing soft rot on potato tubers in Southern Europe

Soft-rot Enterobacteriaceae are recognized as some of the most important bacterial pathogens in agriculture. Among the species, Pectobacterium (Erwinia) atrosepticum, P. carotovorum subsp. carotovorum, and several Dickeya spp. (E. chrysanthemi) are responsible for most potato soft rot in fields worldwide. These pathogens are routinely identified with standard biochemical and physiological tests in combination with PCR assays (Czajkowski et al. 2015). Among the strains isolated from tubers exhibiting symptoms of soft rot during the growing seasons of 2009 to 2016 in Castile and Leon (the major potato producing region in Spain), we found a group of 11 strains that yielded PCR amplicons of the expected size for P. c. subsp. carotovorum (∼550 bp; Kang et al. 2003). By contrast, no PCR products were obtained using primer pairs specific for P. atrosepticum or Dickeya spp. (Peters et al. 2007). However, four of these isolates (CRD 21, CRD 140, CRD 149, and CRD 274) could not be conclusively assigned to P. c. subsp. carotovorum as they all failed to grow at 37°C and in 5% NaCl. This growth behavior, together with the fact that the PCR assay used to identify P. c. subsp. carotovorum is known to also generate products of a similar size in P. wasabiae strains, led us to evaluate these isolates by qPCR using P. wasabiae-specific primers based on the YD repeat protein gene (Kim et al. 2012). In this assay, all four isolates tested positive, indicating that they could be tentatively assigned to P. wasabiae. Nevertheless, a recently published work shows that the potato isolates of P. wasabiae constituted a separated clade from the original type strain P. wasabiae SR91T obtained from horseradish in Japan and have been reclassified into the new species P. parmentieri (Khayi et al. 2016). To confirm the identity of the four isolates obtained in our study, the complete 16S ribosomal RNA gene was sequenced (GenBank accessions KY070302-05) and compared with other sequences in the Ez-taxon database (www.ezbiocloud.net). High sequence identity, 99.6 to 100%, was obtained among these four isolates and the type strain P. parmentieri RNS 08-42-1A, and confirmed they belonged to this species. Additional biochemical characterization revealed that all four isolates resembled the type strain P. parmentieri RNS 08-42-1A in their ability to produce acid from melibiose, raffinose, lactose, and D-galactose. These tests are able to distinguish P. parmentieri RNS 08-42-1A from P. wasabiae SR91 (Khayi et al. 2016). In a pathogenicity test using at least two wound-inoculated potato tubers (10 CFU/ml), the four isolates under study exhibited maceration ability, and caused soft rot lesions about 2 cm in diameter around the point of inoculation in a 48-h period (24°C and 90 to 100% RH). From these tubers, the bacteria were reisolated into pure culture and tested again by qPCR (Kim et al. 2012). In Europe, P. wasabiae strains infecting potato (= P. parmentieri) have only recently been reported in northwestern countries. Nevertheless, it is thought that its worldwide occurrence and geographical distribution may have been underestimated due to misidentifications caused by the phenotypic diversity of this pathogen and/or the limited tools available in the past for its identification. Here, we report for the first time that P. parmentieri is present in Spain and is a causal agent of soft rot in potato fields. Hence, this pathogen should be taken into account for the correct identification and management of this disease.Peer Reviewe