First Report of Pectobacterium versatile Causing Diseases on Potato in Xinjiang Uygur Autonomous Region and Heilongjiang Province, China.

Abstract

Pectobacterium spp. and Dickeya spp. cause aerial stem rot on potatoes worldwide (Charkowski, 2018). Potato plants (cv. Xisen6# or Youjin) with aerial stem rot or blackleg symptoms (Fig. S1) were observed in the commercial fields in Changji, Xinjiang Province in September 2021 and Harbin, Heilongjiang Province in August 2021, in China. The field disease incidences were 45-50% and 15-20% in Changji (2 ha) and Harbin (1 ha), respectively. Five diseased plants from each field site were collected to isolate the pathogen. Symptomatic stems were soaked in 75% ethanol for 2 min, rinsed, and ground in sterile distilled water (Handique et al. 2022). The suspension was plated onto a crystal violet pectate agar (CVP) plate (Ge et al. 2018). Three days after incubation at 28°C, bacterial colonies that developed pits on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al. 2002). Amplified 16S rDNA sequences from two isolates designated as ZRIMU1267 and ZRIMU1366 showed more than 99% sequence identity to P. versatile CFBP6051T type strain and the sequences were submitted to GenBank (accession numbers: OP476349, OP476350). Additionally, six housekeeping genes sequences were uploaded to GenBank: proA (OP487826, OP487832), gyrA (OP487828, OP487834), icdA (OP487823, OP487829), mdh (OP487825, OP487831), gapA (OP487824, OP487830), and rpoS (OP487827, OP487833) (Ma et al. 2007; Waleron et al. 2008). A phylogenetic tree based on concatenated sequences (MLSA) of the housekeeping genes (Fig. S2) of the two isolates was constructed using MEGA X (Tamura et al. 2013).The phylogenetic tree of MLSA sequences shows that the sequences from isolates ZRIMU1267 and ZRIMU1366 clustered with P. versatile CFBP6051T indicating that these isolates are P. versatile at the species level. Koch's postulate were performed on 3-week-old potato seedlings (cv. Favorita) and tubers. The bacterial suspension (100 μl, 105 CFU/ml) or sterile phosphate-buffered solution was injected into the crown area of the seedlings for the development of aerial stem rot or drenched in the potting mix for the development of blackleg, and the plants were covered with polybags to keep 100% humidity at 25° for 2 days. Five seedlings were inoculated for each strain and the experiment was repeated twice. Seven days after stem injection, the infected area of the inoculated seedlings was rotten, turned black, or even lodged, while the controls were symptomless (Fig. S3a). Four days after drench application, the seedlings were wilted and lodged, while the controls were symptomless (Fig. S3 c). Tuber slice assay for soft rot development was performed by adding bacterial suspension (100 μl, 105 CFU/ml). One day after inoculation, the infected tubers rotted, while the controls were symptomless (Fig. S3 b). ZRIMU1267 and ZRIMU1366 were reisolated from infected tissues on CVP plates and identified by 16S rRNA sequences to complete Koch's postulate. Diseases on potato has been reported to be caused by P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense in China (Zhao et al. 2018; Cao et al. 2021; Wang et al. 2021; Handique et al. 2022). P. versatile causing aerial stem rot on potatoes have been reported in Hebei province (Han et al. 2022), while our study reports P. versatile strains that are able to cause multiple diseases on potatoes in Xinjiang Uygur Autonomous Region and Heilongjiang provinces of China. The results indicate that P. versatile might be widely distributed in northern China, and it is necessary to include cropping season and post-harvest strategies to control diseases caused by P. versatile.