First Report of Pectobacterium parmentieri Causing Stem Rot Disease of Potato in Russia

Abstract

Bacterial (aerial) stem rot of potato (Solanum tuberosum) in Russia is caused by the pectolytic bacteria Pectobacterium carotovorum subsp. carotovorum, P. atrosepticum, or Dickeya spp. (De Boer and Kelman 2001). In 2011 to 2014, over 100 pectinolytic enterobacterial isolates were obtained from green potato plants with blackleg symptoms found in four districts (Odintsovo, Dmitrov, Kashira, and Ruza) of Moscow region in severely infected commercial fields planted with cultivars Nevskii, Gala, and Lady Clear. Disease incidence was from 1 to 10% of infected plants in the field in July to August. From 5 to 15 samples were assayed for each of eight evaluated fields. Six bacterial strains isolated during the survey exhibited pectolytic ability on crystal violet pectate (CVP) agar and potato slices, and failed to grow at 37°C, but physiological tests did not conclusively distinguish the bacterium as P. atrosepticum (Khayi et al. 2016). They were identified as Pectobacterium parmentieri (formerly P. wasabiae group isolated from potato plants) (Kornev et al. 2012) based on their inability to elicit hypersensitive reaction on Nicotiana tabacum and their ability to utilize raffinose and lactose. These bacterial strains were gram negative, rod shaped, N-acetylglucosaminyl transferase, gelatin liquefaction, and ONPG-positive and positive for acid production from d-galactose, lactose, melibiose, raffinose, citrate, and trehalose. All strains were negative for indole production, phosphatase activity, reducing sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-methyl-d-glucoside, and d-arabitol. All the strains exhibited pectolytic activity on potato slices. Reference strains of P. carotovorum NCPPB 312, P. atrosepticum NCPPB 549, and P. parmentieri LMG 29774 obtained from international collections were used as controls. Molecular identification of the bacterium was performed with 16S rRNA and recombinase A (recA) coding sequences as previously described (Waleron et al. 2002). BLAST analysis of the 16S rRNA DNA sequence for the strain F148 (syn. PB20) (GenBank accession no. MG493237.1) revealed 99% identity to the 16S rRNA of P. parmentieri WPP163 (Kornev et al. 2012). The other five strains had sequence of 16S rRNA identical to F148. Phylogenetic reconstruction using DNA sequences of recA gene from P. parmentieri F009, F034, F035, F127, F148 (PB20), and F149 (GenBank accession nos. MG518488, MG518489, MG518490, MG518491, and MG518492) and other related taxa clustered these bacteria with strain SCRI488 and other P. parmentieri strains, readily distinguishing them from other closely related species of Pectobacterium. Pathogenicity assays were conducted on stems of four young potato plants for each strain. Five-week-old ‘Docker’ potato plants were wound inoculated by injecting 10 μl of a bacterial suspension (10⁸ CFU/ml) into each stem. Plants were exposed to a 72-h incubation period with 90 to 100% relative humidity at 30°C, and lesions were measured. All inoculated plants exhibited stem rot/blackleg symptoms similar to those observed in the field, including brown water-soaked lesions. Symptoms were not observed on water-inoculated controls. The bacteria reisolated from all inoculated stems caused pitting on CVP and exhibited the same morphology as the original culture and, thus, were confirmed as P. parmentieri using 16S rRNA and recA sequences, fulfilling Koch’s postulates. To our knowledge, this is the first report of bacterial stem rot/blackleg of potato caused by P. parmentieri in the Russian Federation. According to results of several independent diagnostic laboratories, P. parmentieri had second place in occurrence on seed potatoes after P. carotovorum subsp. brasiliense (about 20% of the tested seed stocks) in field season 2017.