First report of bacterial wilt caused by <i>Enterobacter mori</i> on <i>Illicium verum</i> in China

Illicium verum (Lida) is an important economic tree species and traditional spice in China. In December 2024, a new bacterial wilt disease was observed on I. verum in a cultivation base in Funing County (23° 8′ N, 106° 5′ E), Wenshan City, Yunnan Province, China. The disease has caused severe damage during cultivation with 19% incidence rates . At the early stage of the illness, the leaves were covered with small water-soaked spots. Later, these spots gradually expanded and symptoms of yellowing and curling appeared. In severe cases, it can cause plant wilting and death. Infected plants were collected from three cultivated fields. The infected leaf tissues were excised, surface sterilized with 1% NaClO for 5 min, 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 (Wu et al. 2025). Sixteen strains were isolated from 10 samples of infected leaf tissues. Colonies were raised and opaque with smooth margins. Six bacterial colonies were randomly selected for Gram staining. They were stained with crystal violet and iodine solution, and then de-colored with 95% ethanol (Becerra SC et al. 2016). The results showed that they were rod-shaped Gram-negative bacteria. For molecular identification, three purified bacterial isolates were selected, and fragments of the 16S rRNA, gyrB, and rpoB genes were separately amplified using previously reported the primer pairs 27F/1492R (Li et al. 2024), gyrB UP-1/gyrB UP-2r (Yamamoto et al. 1995) and rpoB CM81-F/rpoB CM32b-R (Brady et al. 2008). Sequence analysis revealed that the three isolates shared identical sequences for all three genes (16S rRNA, gyrB, and rpoB). Therefore, strain CR2-5 was selected as the representative isolate, and its sequences were deposited in GenBank under accession numbers PZ330312 (16S rRNA), PZ344526 (gyrB), and PZ344527 (rpoB). BLAST analyses of the three gene sequences of strain CR2-5 with the type strain E. mori LMG 25706 (Zhu et al. 2011) revealed high similarities of 99.87% for 16S rRNA (MG846019), 96.63% for gyrB (JX424992), and 100% for rpoB (EU579858).The phylogenetic tree based on the concatenated sequences of the above three genes using neighbor-joining method with MEGA software showed that CR2-5 clustered with E. mori LMG25706 with a 100% bootstrap value. For pathogenicity tests, one representative bacterial strain was selected. Three healthy plants of I. verum were inoculated by dropping 10 μL of the bacterial suspension (108 CFU/ml) directly onto the leaf surface. Sterile water was used as the negative control. The pathogenicity tests were conducted three times with similar results. On inoculated plants, the leaves developed water-soaked spots and curled at 2 days post-inoculation (dpi) and appeared wilted and necrotic at 7 dpi. The symptoms were similar to those observed in field-infected I. verum plants. No symptoms were observed on the plants treated with water. Re-isolated strains had the same morphological characteristics and the same sequences as the original isolates, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of E. mori causing bacterial disease on I. verum in China. The results will help to target the screening of control agents and provide guidance for disease prevention and control.