First report of bacterial soft rot caused by Enterobacter mori affecting host watermelon

In June 2021, bacterial stem rot-like symptoms were observed on the stems and leaves of watermelon (Citrullus lanatus cv. ‘Zaojia’) in Pingyu County, Zhumadian City, Henan Province, China (32.44N 114.24E), which showed brown to dark brown lesions on the stems (Fig. 1A). The stems then became scorched, and the leaves showed necrotic lesions with small water-soaked spots (Fig. 1B). Watermelon is a very important economic plant in this small county, where the watermelon planting area accounts for about 15% of the arable land area. Approximately 2 hectares of ‘Zaojia’ have been investigated, and the disease incidence rates were almost 20~30%, thus, causing severe economic losses. Ten symptomatic watermelon stems and leaves were randomly collected based on the typical symptoms, brought into the Lab and used to isolate the pathogen. Each infected tissue was excised and cut into small pieces (about 5 mm×5 mm) and surface disinfected with 1% NaClO for 3 min. The pieces were then rinsed three times in sterile distilled water (SDW) and dried by airing. These pieces (4-5 pieces per sample) were macerated in 200 μL SDW for 60 s in a sterile mortar and pestle. A volume of 5 μL suspensions of each sample were streaked onto two LB agar plates and incubated for 48 h at 28 °C in the dark. After incubation, the colonies on LB agar plate were small, round, raised, white to cream-colored, and had smooth margins (Fig. 2). Two strains from each plate were selected. The genomic DNA of all 40 strains was extracted using a Bacterial Genomic DNA Extraction Kit D1600 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions. The 16S ribosomal RNA gene (27F:5'-AGA GTT TGA TCC TGG CTC AG-3', /1492R: 5'-CTA CGG CTA CCT TGT TAC GA-3'), and the three housekeeping genes, including gyrB (Trantas et al., 2013), icdA and proA (Ma et al., 2007), were amplified. Sequence analysis showed that 40 strains shared the same sequence, so only one sequence was submitted into GenBanK.The 16s rDNA partial sequences (SUB12134746) shared 100% similarity with E.mori (CP084692.1), and the gyrB (OP676246), icdA (OP676248) and proA (OP676247) genes shared 98.67%, 99.39% and 97.99% homology with those of E. mori (CP084692.1), respectively. Besides, the phylogenetic tree analysis based on multi-housekeeping gene joint gryB-icdA-proA showed that E.mori（OP676246-OP676248- OP676247）from watermelon was culsterd with the E.mori (CP084692.1) from South Korea and E.mori (CP055276.1) from kiwifruit (Fig. 3). Thus, E.mori was confirmed to be the pathogen responsible for bacterial soft rot of watermelon in this study. To confirm the pathogenicity, 15-day-old healthy cv. ‘Zaojia’ watermelon seedlings were inoculated by spraying all the seedlings with a bacterial suspension (1×10 8 CFU mL-1) at an incubation temperature of 28 °C and 70% relative humidity, and sterile distilled liquid LB medium was applied as a negative control treatment. Three times were conducted for the isolate, and each time included nine watermelon plants. After 10 days, only the inoculated cotyledons and leaves with the bacterial suspension showed bacterial leaf spots that resembled those observed on naturally infected watermelon cotyledons and leaves (Fig. 4A-C), whereas the control plants remained asymptomatic (Fig. 4D). Simultaneously, the watermelon stems were inoculated with the bacterium in vitro. Each stem was slightly wounded with a metal sponge and then sprayed with the bacterial suspension (108 CFU mL-1) of each isolate, and the experiment was repeated three times. Water-soaked symptoms were visible on the stems (Fig. 4E), while the control plants remained asymptomatic (Fig. 4F). The strains were then successfully re-isolated and identified by sequence analyses of their 16S ribosomal RNA gene and gyrB, icdA and proA genes. Therefore, the inoculation experiment of the isolatedbacterium fulfilled Koch’s postulates. Previously, E. mori has been reported to cause bacterial wilt on white mulberry (Morus alba L.) (Zhu et al. 2022), peach fruit (Prunus persica) (Ahmad et al. 2021) and kiwifruit (Actinidia deliciosa [A. Chev.] CF Liang et AR Ferguson) (Zhang et al. 2021). To our knowledge, this is the first report of E. mori causing bacterial soft rot on watermelon in world.