First report of Enterobacter ludwigii causing root rot of Rehmannia glutinosa in China

Rehmannia glutinosa Libosch. is an economically important medicinal crop whose tuberous roots are widely used in traditional Chinese medicine. In January 2026, root rot was observed in a commercial field in Wuzhi Town (35.10°N, 113.40°E), Jiaozuo City, Henan Province, China. The disease incidence in the surveyed field was approximately 50%. Affected plants exhibited foliar wilt, brown-to-black root necrosis, root decay, and reduced vigor. Roots and growth-substrate samples were collected from 10 symptomatic and healthy plants. Root segments (2 cm) from lesion margins and healthy roots were surface-disinfested in 0.1% sodium hypochlorite for 5 min, rinsed three times with sterile distilled water, plated on nutrient agar (NA) and potato dextrose agar (PDA), and incubated at 25°C in darkness. PDA was included to recover and monitor other microorganisms associated with diseased tissues, whereas bacterial isolates used in subsequent tests were recovered and purified on NA. After approximately 3 days, morphologically similar bacterial colonies were recovered from 80% of symptomatic roots. Five bacterial isolates were obtained. Pathogenicity assays were conducted on healthy R. glutinosa plants under greenhouse conditions to fulfill Koch's postulates. For each isolate, bacterial suspensions were adjusted to 1 × 10⁸ CFU/mL. Each treatment contained three plants, and the experiment was repeated three times independently. Roots were superficially wounded with a sterile scalpel to create small wounds, and each plant was inoculated with 5 mL of bacterial suspension. Plants treated with sterile nutrient broth and noninoculated plants served as negative controls. Plants were covered with polyethylene bags for 24 to 48 h to maintain high humidity and maintained in a greenhouse at 25 ± 2°C under a 16-h light/8-h dark photoperiod. Ten days after inoculation, plants inoculated with isolate X21 developed root rot and wilt similar to those observed in the field, whereas plants inoculated with the other four isolates and both control groups remained asymptomatic. Bacteria with colony morphology identical to that of X21 were reisolated from symptomatic roots, thereby fulfilling Koch's postulates (Fig. 1). Isolate X21 formed creamy-white colonies on NA after 24 h at 25°C. The partial 16S rRNA gene was amplified with primers 27F/1492R (Kai et al. 2019), and the sequence was deposited in GenBank (PZ212876.1). Because 16S rRNA sequences provide limited resolution within the Enterobacter cloacae complex, partial gyrB (1,008 bp) and rpoB (991 bp) genes were also sequenced. gyrB was amplified with primers gyrB-F (5′-CCSACSGACGTGAAGATG-3′) and gyrB-R (5′-TCCACTGCATSGCGACTTC-3′), and the resulting sequence was deposited in GenBank (PZ707229). rpoB was amplified with primers rpoB-F (5′-AACCAGTTCCGCGTTGGCCTGG-3′) and rpoB-R (5′-CCTGAACAACACGCTCGGA-3′), and the resulting sequence was deposited in GenBank (PZ707228). Sequences from X21 and type or type-material strains of the E. cloacae complex were aligned using MUSCLE v5.3 (Edgar 2022) and trimmed to the regions covered by the X21 sequences. Maximum-likelihood analyses were performed in IQ-TREE v2.4.0 (Minh et al. 2020) using best-fit models selected according to the Bayesian information criterion and 1,000 ultrafast bootstrap replicates. Escherichia coli K-12 MG1655 was used as the outgroup. The 16S rRNA tree placed X21 within the E. cloacae complex but did not reliably resolve it at the species level. In contrast, X21 clustered with the E. ludwigii type strain EN-119T in both the gyrB and rpoB trees, with bootstrap support of 100 and 88%, respectively. An MLSA tree based on concatenated gyrB + rpoB sequences (1,999 aligned positions) placed X21 with E. ludwigii EN-119T with 100% bootstrap support (Fig. 2). These results identified X21 as E. ludwigii. Enterobacter ludwigii was described as a member of the E. cloacae complex (Hoffmann et al. 2005). Previous studies associated Enterobacter aerogenes and unidentified Enterobacter spp. with root rot or wilt of R. glutinosa (Wang et al. 2018; Li et al. 2020). However, to our knowledge, this is the first report of E. ludwigii causing root rot of R. glutinosa in China. This disease may threaten stand establishment and root production, highlighting the need for surveillance and management in R. glutinosa production areas.