First Report of <i>Dickeya fangzhongdai</i> Causing Soft Rot on <i>Hippeastrum striatum</i> in Guangxi, China

Hippeastrum striatum is a popular ornamental plant in the Amaryllidaceae family (Shi et al. 2020). Soft rot symptoms were observed on H. striatum leaves in a nursery of Guangxi bird and flower market in Nanning, Guangxi, China (22.85366°N, 108.36066°E) with a disease incidence of 22% (38 out of 174) in June 2024. Initially, small, water-soaked, and oval lesions were observed on the leaves, which rapidly expanded in a strip shape along the leaves, leading to leaf discoloration, decaying and death of the whole plant. Six soft rotted leaves were surface disinfected with 70% ethanol for 30 s followed by 5% NaClO for 2 min and rinsed three times with sterile water. Symptomatic leaf tissue was cut into 5x5 mm slices, which were then soaked in 2 ml of sterile water for 2 h, and 100 µl were spread onto potato dextrose agar (PDA) and Luria-Bertani (LB) agar plates after gradient dilutions and incubated at 28°C for 48 h. Colonies from all the six samples appeared as yellowish-white, featuring irregular margins and smooth surfaces and 12 individual colonies were purified by plate streaking method. Two representative strains, ZDH2 and ZDH4, were inoculated on separate Biolog GEN III MicroPlate (Biolog Inc, Hayward, CA), micro sugar fermentation tubes at 30°C for 24 h, and casein biochemical medium at 30°C for a week. Overall, biochemical test results were in accordance with the general characteristics of Dickeya fangzhongdai by growth at 39℃, utilizing arabinose, gentiobiose, D-melibiose, D-mannitol, D-raffinose and myo-inositol, while does not utilize casein, L-rhamnose, tween 40 and N-acetyl-D-galactosamine (Tian et al. 2016; Alič et al. 2018). Amplification of an expected 76 bp amplicon length was obtained for ZDH2 and ZDH4 strains with D. fangzhongdai specific real-time PCR assay Df-trF/R (Alič et al. 2024). The 16S rRNA gene sequences of the two isolates were amplified using primers F27 and R1492 (Heuer et al. 1997). BLASTn analysis showed 99.7% identity and 100% coverage with Dickeya fangzhongdai strain JS5T for both strains. Five housekeeping genes dnaX, fusA, gapA, purA, and recA were also sequenced from ZDH2 and ZDH4 DNA extracts (Tian et al. 2016). The above gene sequences of both strains were submitted to GenBank (16S rRNA: PQ835808, PQ835809, dnaX: PQ842614, PQ842615, fusA: PQ878468, PQ878469, gapA: PQ842616, PQ842617, purA: PQ842618, PQ842619, and recA: PQ842620, PQ842621). A maximum-likelihood phylogenetic analysis was performed on concatenated sequences from the six multi-locus regions using RAxML version 8.2.10. The two isolates form a well-supported clade (100% bootstrap) with D. fangzhongdai JS5T and QZH3. Bacterial suspensions of ZDH2 and ZDH4 were adjusted to 1×108 CFU/ml. For each isolate, five healthy leaves from two H. striatum plants were inoculated by placing 10 µl droplets of the bacterial suspension onto the unwounded adaxial surface; plants were then enclosed in transparent plastic bags to maintain high humidity. Sterile water (10 μL) was used as a negative control. After 48h at 28℃, soft rot symptoms were observed on all inoculated leaves, whereas controls remained symptomless. D. fangzhongdai was successfully reisolated from the symptomatic leaves and confirmed by morphology and 16S rRNA sequence, whereas no pathogen was isolated from mock inoculated controls, fulfilling Koch’s postulates. D. fangzhongdai is known to cause soft rot and bleeding canker in various crops and ornamentals, including Asian pear, onion, banana, taro and orchids, among others in China or other countries (Alič et al. 2024). This study represents the first report of D. fangzhongdai causing soft rot of H. striatum, highlighting its expanding host range and potential threat to ornamental plants. The finding will help to understand the epidemiology and develop management of horticultural plant disease in the future.