First Report of Bacterial Wilt Caused by Enterobacter cloacae on Pogostemon cablin in China

Pogostemon cablin (Blanco) Benth., a key botanical source for numerous traditional Chinese medicinal formulations, experienced severe bacterial wilt outbreaks in June 2023 across commercial plantations in Yunfu City, Guangdong Province. Initial symptoms presented as leaf wilting with water-soaked appearance while retaining green pigmentation, followed by progressive necrosis leading to complete plant death, resulting in approximately 50% yield reduction across affected fields. Symptomatic plants exhibiting vascular discoloration were collected for pathogen isolation. Infected rhizomes were surface-sterilized by immersion in 75% ethanol (30 s) and 2.5% sodium hypochlorite (10 s), rinsed thrice with sterile water, then soaked in sterile water for 12 h. The soaking solution was streaked on LB agar and incubated at 28°C for 24-48 h. From the isolated strains, three exhibited identical colony morphology and were identified as Enterobacter cloacae through 16S rRNA sequencing (99.64% similarity). One representative strain GHXBW was chosen for further investigation. Gram staining confirmed the Gram-negative nature of the pathogen. Biochemical testing demonstrated that strain GHXBW fermented glucose (acid/gas), maltose, mannitol, and sucrose but not lactose; was positive for Voges-Proskauer reaction, urease activity, Simmons' citrate utilization, and motility (semi-solid agar); and negative for methyl red reaction, indole production (peptone water), and H 2 S generation. Subsequent multilocus sequence analysis targeting atpD, gyrB, infB, and rpoB (Brady et al., 2008) genes revealed that strain GHXBW formed a monophyletic clade with E. cloacae DSM 30054 (NZ_CP056776) and E. cloacae FDAARGOS 1431 (NZ_CP077211) with 100% bootstrap support. The nucleotide sequences of atpD, gyrB, infB, and rpoB from strain GHXBW have been deposited in GenBank with accession numbers PV580084 (atpD), PV580085 (gyrB), PV580086 (infB) and PV580087 (rpoB). Pathogenicity was confirmed through root inoculation of 60 days old plants using bacterial suspension (OD 600 = 0.6), which induced leaf wilting within 7 days post-inoculation under controlled conditions (28°C, 80% RH, 14h photoperiod) in a growth chamber, while water-treated controls remained asymptomatic. A total of 12 plants were inoculated (4 plants per trial), of which 7 developed wilt symptoms consistent with those observed under field conditions (3/4, 2/4, and 2/4 plants affected in trials 1-3, respectively). Re-isolation from diseased tissues of all infected plants reproduced identical colonies, with 16S rRNA sequencing verifying E. cloacae identity. In contrast, no E. cloacae colonies were isolated from control plants (water-treated). This pathogen has been previously associated with bacterial wilt in mulberry (Wang et al., 2008), garlic bulb rot (Li et al., 2021), ginger rhizome rot (Zhao et al., 2022),and tomato pith necrosis (Guo et al., 2023) in China, but has never been reported on P. cablin. This is the first report of E. cloacae as the causal agent of bacterial wilt in this medicinal crop. As Guangdong Province accounts for the majority of P. cablin production in China, these results inform the development of targeted phytopathological management strategies in this key growing region.