First Report of Soft Rot Caused by Pectobacterium polaris on Lettuce in China.

Abstract

Lettuce (Lactuca sativa) is a leafy vegetable that belongs to the family Asteraceae. It is widely cultivated and consumed around the world. In May 2022, lettuce plants (cv. 204) showing soft rot symptoms were observed in greenhouses in Fuhai District (25°18'N, 103°6'E), Kunming City, Yunnan Province, China. The disease incidence in three greenhouses (0.3 ha in size) was between 10% to 15%. The lower parts of the outer leaves showed brown and water-soaked symptoms, but at the same time the roots were asymptomatic. Sclerotinia species can cause soft decay on lettuce leaves, known as lettuce drop, which can produce symptoms partially resembling those of bacterial soft rot (Subbarao 1998). The absence of white mycelium or black sclerotia on the leaf surfaces of diseased plants indicated that Sclerotinia species were not responsible for the disease. Instead, it is more likely that bacterial pathogens were the cause. Fourteen diseased plants were sampled from three greenhouses, and potential pathogens were isolated from the leaf tissues of six plant individuals. Leaf samples were cut into pieces ca. 0.5 cm in length. The pieces were then surface-sterilized by dipping in 75% ethanol for 60 sec, followed by three successive rinses using sterile distilled water. The tissues were immersed in 250 µl of 0.9% saline in 2 mL microcentrifuge tubes and gently pressed down with grinding pestles for 10 sec. The tubes were let stand still for 20 min. Aliquots (20 μl) 100-fold dilutions of the tissue suspensions were plated onto Luria-Bertani (LB) plates and incubated at 28°C for 24 h. Three single colonies were picked from each LB plate and restreaked five times for purity. After purification, eighteen strains were obtained, and nine of these were identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991). Six out of nine strains (6/9) belonged to the genus Pectobacterium (OP968950-OP968952, OQ568892- OQ568894), two strains (2/9) belonged to the genus Pantoea (OQ568895 and OQ568896), and one strain (1/9) belonged to Pseudomonas sp. (OQ568897). Since the Pectobacterium strains shared identical 16S rDNA sequence, strains CM22112 (OP968950), CM22113 (OP968951) and CM22132 (OP968952) were selected as representative strains for further testing. The 16S rDNA sequences of Pectobacterium strains were 100% identical to that of the P. polaris strain NIBIO 1392 (NR_159086.1). To identify the strains to the species level, multilocus sequence analysis (MLSA) was performed using sequences of six housekeeping genes acnA, gapA, icdA, mdh, proA and rpoS (OP972517-OP972534) (Ma et al. 2007; Waleron et al. 2008). Phylogenetic analysis showed that the strains clustered with P. polaris type strain NIBIO1006T (Dees et al. 2017). They were all capable of utilizing citrate, which is an important biochemical feature in distinguishing P. polaris from its most closely related sister species P. parvum (Pasanen et al. 2020). Lettuce plants (cv. 204), at the rosette stage, were inoculated with the strains CM22112 and CM22132 by injecting 100 µl of bacterial suspensions (107 CFU·mL-1) into the lower parts of the leaf; for the controls, 100 µl of saline was used instead. Inoculated plants were incubated at room temperature (23°C) and 90% relative humidity. Five days after inoculation, only the bacteria-inoculated lettuce showed severe soft rot symptoms. Similar results were observed in two independent experiments. Bacterial colonies were obtained from the infected lettuce leaves, which showed identical sequences to P. polaris strains CM22112 and CM22132. Therefore, these strains fulfilled Koch's postulates for lettuce soft rot. P. polaris is prevalent on potato in many countries (Dees et al. 2017). To our knowledge, this is the first report of P. polaris causing soft rot on lettuce in China. This disease could seriously affect the appearance and saleability of lettuce. Further research on the epidemiology and management strategies of the disease is needed.