First Report of Enterobacter kobei and Enterobacter mori Causing Ginger Bacterial Wilt in China

Ginger (Zingiber officinale) is an important medicinal herb and vegetable in China. In August 2023, a severe outbreak of bacterial wilt of ginger plants (Tongling White Ginger) was observed in Yi'an District (30°45'N, 117°43'E) in Tongling City, Anhui Province, China. There were over 1,000 acres of Tongling white ginger, and the disease incidence rate in the field exceeded 30%. Symptomatic plants displayed browning and softening at the stem base. These symptoms resembled those caused by Ralstonia solanacearum and Enterobacter cloacae, known causal agents of bacterial wilt (Liu et al. 2021; Yu et al. 2003), suggesting that diverse pathogens might be involved in ginger bacterial wilt. To identify the causative agents, symptomatic plants were harvested from various ginger-cultivated fields. Ten symptomatic plants were randomly sampled from different locations, and decayed stem tissues were excised, surface-sterilized with 1% NaClO for 10 minutes, rinsed with sterile water, and ground. Serial dilutions of the supernatant were plated on nutrient agar (NA) medium and incubated at 28°C for 24 h. The 16S rRNA genes of purified isolates were amplified using primers 27F/1492R and sequenced (Weisburg et al. 1991). Taxonomic identification via BLASTn revealed that 56 isolates belonged to the genus Enterobacter. To further classify these isolates at the species level, the hsp60 gene was amplified (Hoffmann et al. 2003). Five isolates were identified as Enterobacter kobei, with 100% sequence similarity to E. kobei C5 (MN962751, 267/267 bp), and three isolates were identified as Enterobacter mori, with 100% sequence similarity to E. mori CX01 (CP055276, 294/294 bp). Specific primers for four housekeeping genes including gyrB, rpoB, atpD, and infB were also used for PCR amplification (Brady et al. 2013). Sequence analysis showed that all five E. kobei isolates shared identical sequences, matching E. kobei EK9 (MZ325275, 495/495 bp; MZ325277, 615/615 bp; MZ325278, 603/603 bp; MZ325276, 657/657 bp) at 100% similarity across these genes. Moreover, the three E. mori isolates were identical, with nucleotide sequence similarities of 97.19%, 99.21%, 99.67%, and 98.37% for gyrB, rpoB, atpD, and infB, respectively, to E. mori LMG 25706 (JX424992, 622/640 bp; JX425251, 625/630 bp; JX424862, 604/606 bp; JX425121, 602/612 bp). A representative isolate from each species, designated E. kobei EK02 and E. mori EM07, was selected, and gene sequences for hsp60 (PQ566999 and PQ567000), gyrB (PQ566991 and PQ566992), rpoB (PQ566993 and PQ566994), atpD (PQ566995 and PQ566996), and infB (PQ566997 and PQ566998) were deposited in the GenBank database, respectively. Phylogenetic trees were constructed based on concatenated sequences of gyrB, rpoB, atpD, and infB, as well as hsp60, using the Neighbor-Joining method in MEGA (Alvarez et al. 2018). These phylogenetic analyses confirmed that EK02 clustered with E. kobei and EM07 clustered with E. mori. To confirm pathogenicity, EK02 and EM07 were cultured on NA liquid medium at 28°C, then resuspended in sterile water to a final concentration of 108 CFU/ml. A 10 µl aliquot of the bacterial suspension was injected into the stem base of two-month-old ginger plants, with sterile water serving as the negative control. Nine plants were inoculated with each isolate, and each treatment was replicated three times. These plants were maintained in a growth chamber at 35°C and 80% relative humidity. Symptoms appeared within five days post-inoculation, whereas control plants remained symptom-free. By sequencing and analyzing the 16S rRNA, hsp60 and housekeeping genes, the pathogens re-isolated from symptomatic tissues were identified as EK02 and EM07, thereby satisfying Koch's hypothesis. To our knowledge, this is the first report of E. kobei and E. mori as causal agents of ginger bacterial wilt in China. The identification of these pathogens is crucial for developing effective management strategies to prevent bacterial wilt outbreaks in ginger plants.