First Report of Pectobacterium carotovorum and Pectobacterium aroidearum Causing Bacterial Soft Rot on Curly Dwarf Pak Choy (Brassica rapa var. Chinensis) in Malaysia

Brassica rapa var. Chinensis (curly dwarf pak choy) is commonly grown in large-scale vertical farming aquaponic systems. In October 2022, soft rot symptoms and dark brown lesions were observed on B. rapa grown in a commercial aquaponic farm located in Perak, Malaysia. The infected stem appeared brown and water soaked. Severely infected plants produced creamy white ooze on the surface before collapsing entirely (Fig. 1A and B). Infected leaves displayed yellow-brown symptoms and eventually rotted (Fig. 1C); the healthy plants were symptomless (Fig. 1D). About 20 % of the 20,000 B. rapa plants on the farm exhibited symptoms. Ten randomly selected symptomatic plants, five with infected stems and five with infected leaves, were surface sterilized. Each tissue (1.0 cm2) was homogenized and suspended in a saline solution. The suspensions were then serially diluted and plated separately on Luria-Bertani agar. After a 16-h incubation period, stem tissue yielded 12 isolated colonies, while leaf tissue produced 8 colonies. These isolates were subjected to dereplication using RAPD-PCR (Krzewinski et al., 2001), revealing two distinct RAPD patterns. The cultures, named Pathogen Stem 2 (PS2, obtained from the stem) and Pathogen Leaf 2 (PL2, obtained from the leaf), were initially identified as Pectobacterium sp. through 16S rRNA sequence analysis (Frank et al., 2008) on the EzBioCloud 16S database (Yoon et al., 2017). Further identification of the Pectobacterium species was conducted using multilocus sequence analysis (MLSA) of the icdA, mdh, proA, and mltD genes (Ma et al., 2007). The sequences were deposited in GenBank (OQ660180, OQ660181, and OR206482-OR206489). Based on MLSA phylogeny, PS2 and PL2 were identified as Pectobacterium carotovorum and Pectobacterium aroidearum, respectively (Fig. 2A). Anaerobic assays confirmed their facultative anaerobic nature, while Gram staining revealed Gram-negative, rod-shaped morphology consistent with Pectobacterium (Fig. 2B and C). For the re-inoculation study, one-month-old healthy B. rapa plants were used. PS2 was inoculated into petioles, while PL2 was inoculated into leaves separately (3 biological replicates × 3 leaves for each replicate) using the prick inoculation method (Wei et al., 2019). Sterile needles were used to prick the plant tissues, and 10 µL of bacterial suspensions (2.40×109 CFU/mL) in saline were inoculated onto the pricked spots. Negative control using sterile saline was included. The inoculated plants were maintained in a controlled growth chamber (25 ± 1°C, relative humidity 80 ± 5%). After 48 hpi, the petiole tissue inoculated with PS2 showed bacterial soft rot symptoms (Fig. 1F) and leaves inoculated with PL2 appeared dark brown around the wound (Fig. 1G), similar to the symptoms observed in the commercial farm (Fig. 1B, C); while control plants remained asymptomatic (Fig. 1E). Bacteria were re-isolated from the inoculated petiole and leaf tissue and their identities were confirmed by RAPD-PCR. The RAPD profiles of the bacteria reisolated from the petiole and leaf tissues were the same as those of PS2 and PL2 respectively (Fig. 1H). The pathogenicity of PS2 and PL2 was thus confirmed. To our knowledge, this is the first report of bacterial soft rot on B. rapa in aquaponic systems caused by P. carotovorum and P. aroidearum in Malaysia. The identification of these pathogens is crucial for the prevention of disease outbreaks and to develop an effective disease management strategy.