Ginger Rhizome Rot Caused by the Enterobacter cloacae in Tangshan, China.

Abstract

Ginger (Zingiber officinale ROSC.) is an important economic crop in China, especially the rhizome tissue has a high medicinal value. In July 2019, the symptom of ginger rhizome rot appeared in Tangshan city of Hebei Province, with incidence rates of 15%. The diseased part of ginger rhizome became soft and presented light brown maceration. Serious internal all erosion, only have epidermis, gray-white juice overflowed the epidermis, and with foul smell. The surface of ginger rhizome was disinfected with 1% NaOCl, and colonies were isolated and purified on nutrient agar (NA) solid medium by streak plate method. Eight isolates were obtained from 15 diseased tissue samples. Further morphological, physiological and biochemical identification of the pure cultured bacteria were carried out. Two isolates of bacteria were picked for further analysis. The bacteria were gram-negative bacilli, which were milky white and round protuberances on NA solid medium. Physiological and biochemical test results showed that isolates were facultatively anaerobic, gelatin liquefaction; negative for indole, methyl red, hydrogen sulfide production and the Voges-Prauskauer test (V-P); positive for D-glucose, sucrose, sorbitol, inositol, mannitol and citrate utilization. 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 24-h. These characteristics were consistent with Enterobacter spp. To further assess the identity of the species, the genomic DNA was extracted from one bacterium（SDXJ1）. The partial 16S rRNA gene and specific rpoB and gyrB genes were amplified and sequenced with primers 27F/1492R, CM7/CM31b and UP1f/UP2r (Brady et al. 2013, Mollet et al. 1997, Lane 1991). The obtained 16S and rpoB sequences (GenBank accession MK937637, MZ911902 and MZ911901) of the isolate showed 99.33%, 99.21% and 99.57% identity to the corresponding sequences of Enterobacter cloacae in GenBank (CP055458, AP022228 and AP022519). Maximum likelihood analysis was performed, and the phylogenetic tree clustered with E. cloacae (MEGAX, Bootstrap n=1000). The pathogenicity of the isolates was tested on ginger plants and rhizomes tissue. The bacterial suspensions (108 CFU/mL) of two isolates were injected into the basal stem and rhizomes center of 6 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. The pathogens of symptomatic plants and ginger rhizomes were studied. They were identified as E. cloacae by physiological, biochemical and molecular biological methods, fulfilling Koch's hypothesis. This is the first report of the ginger rhizome rot caused by the Enterobacter cloacae in Tangshan, China. The research results are of great significance for the prevention and control of the disease. Our laboratory has reported that Citrobacter freundii can cause Ginger Rot. (Zhao et al. 2021) Whether the two strains infect alone or compound in the field still needs to be further explored.