LAUSR

In July 2020, potato plants (cv. Xisen 6) showing characteristic symptoms of aerial stem rot were observed in a field in Fengning Manchu Autonomous County, Chengde, Hebei Province (northern China). The disease incidence in that field (5 ha in size) was more than 50%. Aerial stem rot of potato has increased in prevalence over recent years in Chengde, it can cause significant yield loss on susceptible cultivars such as Xisen 6 and Huangxin 226. Affected stem (light brown and water-soaked stem sections) pieces ca. 0.5 cm in length were surface-sterilized by dipping them in 75% ethanol for one min and then three successive rinses with sterile distilled water. Then, the tissues were soaked in 200 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the crystal violet pectate (CVP) medium. The CVP plates were incubated at 28°C for 48 h. Colonies producing pits were restreaked and purified on Luria-Bertani (LB) agar plates. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R (Weisburg et al. 1991) and sequenced. Results of the Blastn analysis of the 16S rDNA amplicons (MZ348607, MZ348608) suggested that the isolates FN20211 and FN20222 belonged to the genus Pectobacterium. Housekeeping genes including acnA, gapA, icdA, mdh, proA and rpoS were also amplified using a set of primers (Ma et al. 2007; Waleron et al. 2008) followed by sequencing (MZ356250-MZ356261). To determine the species of the stem rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes, and phylogenetic tree was reconstructed using RAxML (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between FN20211 and FN20222. The result of phylogenetic analysis showed that the isolates clustered with P. polaris type strain NIBIO1006T, which was isolated from potato (Dees et al. 2017). And the concatenated sequence of the six loci of isolate FN20211/FN20222 is 100% identical to those of the strains PZ1 (CP046377.1) and WBC1 (GCF_011378945.1), which were isolated from potato in South Korea and from Chinese cabbage in China, respectively. Potato seedlings (cv. Xisen 6 and Favorita) were inoculated with the isolates FN20211 and FN20222 by injecting 100 µl of bacterial suspensions (108 CFU·mL-1) into the upper parts of the stems of potato plants, or injected with 100 µl of 0.9% saline as control. The seedlings were grown at 25°C and 50% relative humidity. Three days after inoculation, only the bacteria-inoculated seedlings showed disease symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected stems and were identified using the same PCR primers as described above. Therefore, P. polaris isolates FN20211 and FN20222 fulfill Koch's postulates for aerial stem rot of potato. P. polaris causing blackleg and soft rot on potato plants has been reported in European countries including Netherlands, Norway (Dees et al. 2017) and Poland (Waleron et al. 2019), and also in Pakistan (Sarfraz et al. 2019) and Russia (Voronina et al. 2021). To our knowledge, this is the first report of P. polaris causing aerial stem rot of potato in China. The stem rot poses a significant threat to the local potato industry, and further research on epidemiology and disease management options is needed.