First Report of <i>Enterobacter dissolvens</i> Causing Ginger Bacterial Wilt in China

Tongling White Ginger (Zingiber officinale), a protected geographical indication product, is a vital economic crop with cultivation spanning over 2,500 years in Yi'an District, Tongling City (30°45'N, 117°43'E) (Hua et al. 2024). In August 2024, severe wilting symptoms were observed in ginger fields, affecting approximately 30% of plants across 500 hectares. Diseased plants exhibited leaf wilting, yellowing, and characteristic brown discoloration of vascular tissues. To identify the causal pathogens, symptomatic rhizomes and stems of diseased plants from multiple locations were surface-sterilized with 70% ethanol, rinsed in sterile water, and small pieces of vascular tissues were macerated in sterile water. Serial dilution of the resulting suspension was spread onto nutrient agar (NA) plates and incubated at 28°C for 48 h. Subsequently, dominant colonies (creamy white, round, and mucoid) were selected and subcultured on NA plates at least three times to obtain pure cultures using the streak plate method. Three of these purified isolates were randomly selected for further analysis. Biochemical assays of these isolates indicated positive reactions for catalase, Voges-Proskauer test, and citrate utilization, while oxidase and indole production were negative. For molecular identification, 16S rRNA genes of isolates ED03, ED12 and ED36 (GenBank accession No. PX062107-PX062109), were amplified and sequenced using primers 27F/1492R (Spilker et al. 2012). BLASTn analysis showed that 16S rRNA gene sequences of the three isolates ED03, ED12 and ED36 exhibited more than 99% similarity with those of Enterobacter cloacae complex strains. To refine species identification, gyrB, rpoB, atpD, infB and fusA were amplified (Wu et al. 2025). Sequence analysis of these housekeeping genes (gyrB: PX088835-PX088837; rpoB: PX088838-PX088840; atpD: PX088841-PX088843; infB: PX088844-PX088846; fusA: PX088847-PX088849) showed high similarity with E. dissolvens strain LMG2683 (Rameshkumar et al. 2010). A multilocus phylogenetic tree was constructed based on the concatenated sequences of gyrB, rpoB, atpD, infB and fusA genes using the neighbor-joining method in MEGA11.0 software with 1,000 bootstrap replicates. The isolates ED03, ED12 and ED36 clustered most closely with E. dissolvens strain LMG2683. Pathogenicity was further tested by inoculating healthy two-month-old ginger seedlings via injection of 10 μl of bacterial suspensions (10⁸ CFU/ml) into the stem base. Control plants were injected with an equal volume of sterile water. There were three replicates per treatment, each containing six plants. After 7 days in a greenhouse at 30°C, inoculated plants developed wilting and vascular browning identical to field symptoms, while controls remained healthy. Bacteria re-isolated from symptomatic plants were confirmed to be E. dissolvens by sequencing the 16S rRNA and housekeeping genes, matching the original isolates both morphologically and molecularly, thus fulfilling Koch’s postulates. This is the first report of E. dissolvens causing bacterial wilt in Tongling White Ginger in China. The emergence of this pathogen represents a significant threat to production, necessitating urgent research into region- and cultivar-specific management strategies.