First Report of <i>Pseudomonas serbiensis</i> Causing Bacterial Midrib Rot of Hydroponically Cultivated Romaine Lettuce in South Korea

In Korea, lettuce is primarily used for wraps; however, recent changes in food culture have led to an increase in the consumption of romaine lettuce (Lactuca sativa var. longifolia) for salads (Chang et al. 2020). In October 2024, romaine midrib rot was observed in hydroponically cultivated romaine lettuce at a farm in Gwangju, Jeollanam-do, South Korea. Approximately 10% of the plants exhibited bacterial midrib rot-like symptoms, caused by Pseudomonas cichorii (Pauwelyn et al. 2010), including black spots around the inner leaves, black stripes on the midrib, and rotting midribs. To isolate the pathogenic agents, diseased rib tissue was surface-sterilized with 70% ethanol for 30 s, rinsed with sterile distilled water (SDW), ground using a disposable polypropylene rod and micro-pestle in 1.5-ml tubes, suspended in SDW, and serially diluted. The dilutions were spread on trypticase soy (TS) agar (Schaad et al. 2001) and incubated at 28°C for 48 h. Cream-colored circular colonies were predominant on the TS agar and exhibited fluorescence on King’s B (KB) agar. Three cream-colored colonies (B3a–c) isolated from different midribs were purified through repeated dilution and spreading until pure, single-organism colonies were isolated on TS agar plates. The isolates were identified by analysis of five housekeeping genes, gltA, rpoD, gapA, gyrB, and 16S rRNA, amplified using the primer pairs, gltA174/gltA1192, PsEG30F/PsEG790R, gapA312/gapA874, UP1E/APrU, and 27mF/1492mR, respectively (Fullem et al. 2024; Zhao et al. 2023). Sequences of gltA, rpoD, gapA, gyrB, and 16S rRNA locus of the three isolates were identical, and the B3a sequences were deposited in GenBank (acc. nos. PX310631, PV935294, PX310632, PV935293, and PV888705, respectively). Concatenated sequences of the four genes in addition to Pseudomonas syringae species complex were used for phylogenetic analysis using the maximum likelihood method in MEGA 11 (Fullem et al. 2024). The romaine lettuce isolates clustered with the Pseudomonas serbiensis type strain KFB 138T (Fullem et al. 2024) and were distinct from P. cichorii; hence, they were identified as P. serbiensis. They showed 97.69%, 97.4%, 97.57%, 97.93%, and 99.93% homology with those of P. serbiensis KFB 138T, respectively. In the LOPAT test (levan production, oxidase activity, pectolytic activity, arginine dihydrolase production, and hypersensitive response on tobacco) (Lelliott et al. 1966), all three romaine isolates were negative for levan production and arginine dihydrolase, and positive for oxidase activity, pectolytic activity, and tobacco hypersensitivity. However, P. serbiensis KFB 138T is pectolytic negative (Fullem et al. 2024). To confirm Koch’s postulates, the leaf midribs of three commercially obtained 30-day-old romaine lettuces (cv. Minicup) were syringe-injected with a suspension (108 CFU/ml) of each isolate. SDW was injected as a negative control. Plants were kept in a closed plastic box at 25°C. Two days post-inoculation, the inoculated midribs turned dark brown with rot extending into the leaves, whereas negative controls appeared unaffected. Furthermore, P. serbiensis was only reisolated from the symptomatic leaves and confirmed by sequencing the 16S rRNA gene. Pathogenicity tests were conducted twice. This is the first report of P. serbiensis causing P. cichorii-like midrib rot in romaine lettuce in South Korea. Further research is needed to determine how bacteria enter hydroponic systems.