Genetic Diversity of Pectobacterium spp. on Potato in Serbia

Abstract

Pectobacterium is a diverse genus which comprises of multiple destructive bacterial species which cause soft rot/blackleg/wilt disease complex in a wide variety of crops by employing high levels of virulence factors. During the 2018, 2019 and 2020 potato growing seasons, numerous outbreaks of bacterial wilt, stem blackleg and tuber soft rot were recorded, and symptomatic plant samples from ten localities in the Province of Vojvodina (Serbia) were collected and analysed. Bacterial soft-rot pathogens were detected in 63 samples using genus and species-specific primers. Through 16S rRNA Sanger sequencing of 19 representative isolates, the identity of P. brasiliense (73.7%), P. punjabense (15.8%), and P. carotovorum (10.5%) species were revealed. To further validate the identification, genotypic profiling of Pectobacterium strains using rep-PCR (ERIC, BOX, REP) was conducted for 25 selected isolates and the phylogenetic assessment based on four selected housekeeping genes (gyrA, recA, rpoA, and rpoS). Physiological and biochemical properties were analysed using basic microbiological tests and VITEK^® 2 GN card, and pathogenicity was confirmed on cv. VR808 and cv. Desiree potato tubers and plants. This study confirmed the distinctiveness of the newly described P. punjabense in Serbia as well as the high diversity of Pectobacterium brasiliense and Pectobacterium carotovorum species in Serbia.

Keywords: MLSA; Pectobacteriaceae; Pectobacterium punjabense; blackleg; genetic diversity; potato; soft rot.

Figure A1

Potato blackleg, aerial stem rot, wilt, chlorosis and tuber soft rot symptoms detected in potato growing fields in the Province of Vojvodina during 2018–2020.

Figure 1

Dendrogram generated using UPGMA clustering method based on (a) ERIC-PCR, (b) BOX-PCR and (c) REP-PCR results for 21 isolates from Serbia and four reference strains. Genetic distances are described with numbers placed on the branches. Dendrograms were generated using PyElph version 1.3 (Python Software Foundation^®, Wilmington, DE, USA).

Figure 1

Dendrogram generated using UPGMA clustering method based on (a) ERIC-PCR, (b) BOX-PCR and (c) REP-PCR results for 21 isolates from Serbia and four reference strains. Genetic distances are described with numbers placed on the branches. Dendrograms were generated using PyElph version 1.3 (Python Software Foundation^®, Wilmington, DE, USA).

Figure 2

Multilocus sequence analysis of concatenated partial sequences of four housekeeping genes gyrA, recA, rpoA and rpoS of 13 Pectobacterium strains. Maximum likelihood tree built using MEGA X. D. dianthicola strains NCPPB 453 and 16LI02 served as an outgroup. Bootstrap values were calculated using 1000 replicates.

Figure 3

Results of the VITEK GN assay with the panel of Pectobacterium species. Strains 1—P. carotovorum strain MMDC11, 2—P. brasiliense strain MMKC19, 3—P. brasiliense strain MMSGC1, 4—P. punjabense strain MMZKMVR1, 5—P. punjabense strain MMZCVR2, 6—P. carotovorum strain DAPP-PG 751, 7—P. brasiliense strain LMG 21370.

Figure 4

Potato tuber with soft rot registered 48 h post inoculation on cv. Desiree and VR808 inoculated with (a)-P. brasiliense strain MMKC19, (b)-P. carotovorum strain MMDC11, (c)-P. punjabense strain MMZCVR2, (d)-P. carotovorum strain DAPP-PG 752, (e)-P. brasiliense strain LMG 21370, (f)-negative control (left row—longitudinal section of the inoculated tuber; right row—tuber after the removal of macerated tissue).

Figure 5

Blackleg and stem wilt symptoms registered 48 h post inoculation on cv. Desiree and cv. VR808 inoculated with (a)-P. brasiliense strain MMKC19, (b)-P. carotovorum strain MMDC11, (c)-P. punjabense strain MMZCVR2, (d)-P. carotovorum strain DAPP-PG 752, (e)-P. brasiliense strain LMG 21370, (f)-negative control.