Ginger (Zingiber officinale Rosc.) is an important economic crop and its rhizome can be used as seasoning agent and traditional medicine in China. During July 2018 and 2019, decay symptoms occurred in the ginger planting area of Tangshan City, Hebei Province, with incidence rates of 15%～20%. The pathogen infected the rhizomes and leaves. The symptoms included leaves chlorosis and gradually wilting, even the whole plant wilted, the rhizome became soft and presented light brown maceration. In serious cases, the interior of rhizome was completely eroded, gray-white juice overflowing the epidermis, and with foul smell. The rhizome surfaces of ginger plants were disinfected with 1% NaOCl, and colonies were isolated and purified on nutrient agar (NA) solid medium by streaking. Eight isolates were obtained from 15 diseased tissue samples. Further morphological, physiological and biochemical identification of the pure cultured bacteria were carried out. Three strains of bacteria were picked for further analysis. All of the three strains were gram-negative, short rod-shaped，nonmotile bacillus. Colonies were round and milky yellow, smooth raised, and moist after incubation at 28°C for 24h on NA. Physiological and biochemical test results showed that strains were facultatively anaerobic, negative for indole, methyl red, the Voges-Prauskauer test (V-P) and urease; positive for glucose, sucrose, sorbitol, inositol, mannitol, citrate utilization and hydrogen sulfide production; gelatin liquefaction. A typical hypersensitive reaction was induced on 12-week-old tobacco (Nicotiana benthamiana) leaves, which were inoculated by injecting suspensions of the isolated strain (108 CFU/mL) at 25 ℃ after 24h. These characteristics were consistent with Citrobacter freundii (Werkman and Gillen 1932). To further assess the identity of the strains, the genomic DNA was extracted from one bacterium（JXJ4）. The partial 16S rRNA region (Lane 1991) and specific rpoB and gyrB genes (Mollet et al. 1997, Brady et al. 2013) were amplified and sequenced with primers 27F/1492R, CM7/CM31b and UP1f/UP2r, respectively. The obtained 16S, rpoB and gyrB sequences (GenBank accession MN148645, MN158728 and MW199734) of the isolate showed 99.93%, 99.51% and 99.82% identity to the corresponding sequences of C. freundii in GenBank (CP024679.1, CP024677.1 and KM509081.1). Maximum likelihood analysis was performed, and the phylogenetic tree clustered with C. freundii (MEGAX, Bootstrap n=1000). The pathogenicity of the isolates was tested on ginger plants and rhizomes tissue. The bacterial suspensions (108 CFU/mL) of three isolates were injected into the basal stem and rhizomes center of 9 healthy ginger seedlings respectively, and Control groups were treated with sterile water. The inoculated plants were kept in a moist chamber (28°C, 16-h light and 8-h dark period) and ginger rhizomes were placed in the incubator (30°C, 16-h light and 8-h dark period). Seven days after inoculation, the ginger tubers showed symptoms of decay, and 20 to 25 days later, the ginger plant leaves browned and died. The pathogenicity test was repeated 4 times and all controls were healthy. Pathogens were reisolated from symptomatic plants and rhizomes and identified as C. freundii based on the morphological, biochemical and molecular methods described previously, fulfilling Koch's hypothesis. To our knowledge, this is the first report of ginger rot caused by C. freundii in China.
Keywords: Citrobacter freundii; Pathogen detection; Subject Areas; Zingiber officinale Rosc..